Lipid formulations

ABSTRACT

The invention features a cationic lipid of formula I, 
     
       
         
         
             
             
         
       
         
         
           
             an improved lipid formulation comprising a cationic lipid of formula I and corresponding methods of use. 
           
         
       
    
     Also disclosed are targeting lipids, and specific lipid formulations comprising such targeting lipids.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.13/357,856, filed Jan. 25, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/813,448, filed Jun. 10, 2010, which issued asU.S. Pat. No. 8,158,601, which claims priority to U.S. PatentApplication No. 61/185,800, filed Jun. 10, 2009 and U.S. PatentApplication No. 61/244,834, filed Sep. 22, 2009, the contents of each ofwhich are incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 16, 2014, isnamed 08050.014US3_SL.txt and is 27,209 bytes in size.

TECHNICAL FIELD

The invention relates to the field of therapeutic agent delivery usinglipid particles. In particular, the invention provides cationic lipidsand lipid particles comprising these lipids, which are advantageous forthe in vivo delivery of nucleic acids, as well as nucleic acid-lipidparticle compositions suitable for in vivo therapeutic use.Additionally, the invention provides methods of preparing thesecompositions, as well as methods of introducing nucleic acids into cellsusing these compositions, e.g., for the treatment of various diseaseconditions.

DESCRIPTION OF THE RELATED ART

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, andimmune stimulating nucleic acids. These nucleic acids act via a varietyof mechanisms. In the case of siRNA or miRNA, these nucleic acids candown-regulate intracellular levels of specific proteins through aprocess termed RNA interference (RNAi). Following introduction of siRNAor miRNA into the cell cytoplasm, these double-stranded RNA constructscan bind to a protein termed RISC. The sense strand of the siRNA ormiRNA is displaced from the RISC complex providing a template withinRISC that can recognize and bind mRNA with a complementary sequence tothat of the bound siRNA or miRNA. Having bound the complementary mRNAthe RISC complex cleaves the mRNA and releases the cleaved strands. RNAican provide down-regulation of specific proteins by targeting specificdestruction of the corresponding mRNA that encodes for proteinsynthesis.

The therapeutic applications of RNAi are extremely broad, since siRNAand miRNA constructs can be synthesized with any nucleotide sequencedirected against a target protein. To date, siRNA constructs have shownthe ability to specifically down-regulate target proteins in both invitro and in vivo models. In addition, siRNA constructs are currentlybeing evaluated in clinical studies.

However, two problems currently faced by siRNA or miRNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as the freesiRNA or miRNA. These double-stranded constructs can be stabilized byincorporation of chemically modified nucleotide linkers within themolecule, for example, phosphothioate groups. However, these chemicalmodifications provide only limited protection from nuclease digestionand may decrease the activity of the construct. Intracellular deliveryof siRNA or miRNA can be facilitated by use of carrier systems such aspolymers, cationic liposomes or by chemical modification of theconstruct, for example by the covalent attachment of cholesterolmolecules. However, improved delivery systems are required to increasethe potency of siRNA and miRNA molecules and reduce or eliminate therequirement for chemical modification.

Antisense oligonucleotides and ribozymes can also inhibit mRNAtranslation into protein. In the case of antisense constructs, thesesingle stranded deoxynucleic acids have a complementary sequence to thatof the target protein mRNA and can bind to the mRNA by Watson-Crick basepairing. This binding either prevents translation of the target mRNAand/or triggers RNase H degradation of the mRNA transcripts.Consequently, antisense oligonucleotides have tremendous potential forspecificity of action (i.e., down-regulation of a specificdisease-related protein). To date, these compounds have shown promise inseveral in vitro and in vivo models, including models of inflammatorydisease, cancer, and HIV (reviewed in Agrawal, Trends in Biotech.14:376-387 (1996)). Antisense can also affect cellular activity byhybridizing specifically with chromosomal DNA. Advanced human clinicalassessments of several antisense drugs are currently underway. Targetsfor these drugs include the bcl2 and apolipoprotein B genes and mRNAproducts.

Immune-stimulating nucleic acids include deoxyribonucleic acids andribonucleic acids. In the case of deoxyribonucleic acids, certainsequences or motifs have been shown to illicit immune stimulation inmammals. These sequences or motifs include the CpG motif,pyrimidine-rich sequences and palindromic sequences. It is believed thatthe CpG motif in deoxyribonucleic acids is specifically recognized by anendosomal receptor, toll-like receptor 9 (TLR-9), which then triggersboth the innate and acquired immune stimulation pathway. Certain immunestimulating ribonucleic acid sequences have also been reported. It isbelieved that these RNA sequences trigger immune activation by bindingto toll-like receptors 6 and 7 (TLR-6 and TLR-7). In addition,double-stranded RNA is also reported to be immune stimulating and isbelieve to activate via binding to TLR-3. One well known problem withthe use of therapeutic nucleic acids relates to the stability of thephosphodiester internucleotide linkage and the susceptibility of thislinker to nucleases. The presence of exonucleases and endonucleases inserum results in the rapid digestion of nucleic acids possessingphosphodiester linkers and, hence, therapeutic nucleic acids can havevery short half-lives in the presence of serum or within cells.(Zelphati, O., et al., Antisense. Res. Dev. 3:323-338 (1993); andThierry, A. R., et al., pp 147-161 in Gene Regulation: Biology ofAntisense RNA and DNA (Eds. Erickson, R P and Izant, J G; Raven Press,NY (1992)). Therapeutic nucleic acid being currently being developed donot employ the basic phosphodiester chemistry found in natural nucleicacids, because of these and other known problems.

This problem has been partially overcome by chemical modifications thatreduce serum or intracellular degradation. Modifications have beentested at the internucleotide phosphodiester bridge (e.g., usingphosphorothioate, methylphosphonate or phosphoramidate linkages), at thenucleotide base (e.g., 5-propynyl-pyrimidines), or at the sugar (e.g.,2′-modified sugars) (Uhlmann E., et al. Antisense: ChemicalModifications. Encyclopedia of Cancer, Vol. X., pp 64-81 Academic PressInc. (1997)). Others have attempted to improve stability using 2′-5′sugar linkages (see, e.g., U.S. Pat. No. 5,532,130). Other changes havebeen attempted. However, none of these solutions have proven entirelysatisfactory, and in vivo free therapeutic nucleic acids still have onlylimited efficacy.

In addition, as noted above relating to siRNA and miRNA, problems remainwith the limited ability of therapeutic nucleic acids to cross cellularmembranes (see, Vlassov, et al., Biochim. Biophys. Acta 1197:95-1082(1994)) and in the problems associated with systemic toxicity, such ascomplement-mediated anaphylaxis, altered coagulatory properties, andcytopenia (Galbraith, et al., Antisense Nucl. Acid Drug Des. 4:201-206(1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. In Zelphati, O and Szoka, F. C., J. Contr.Rel. 41:99-119 (1996), the authors refer to the use of anionic(conventional) liposomes, pH sensitive liposomes, immunoliposomes,fusogenic liposomes, and cationic lipid/antisense aggregates. SimilarlysiRNA has been administered systemically in cationic liposomes, andthese nucleic acid-lipid particles have been reported to provideimproved down-regulation of target proteins in mammals includingnon-human primates (Zimmermann et al., Nature 441: 111-114 (2006)). Inspite of recent progress, there remains a need in the art for improvedlipid-therapeutic nucleic acid compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high-efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these lipid-nucleic acid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient. The invention provides suchcompositions, methods of making the compositions, and methods of usingthe compositions to introduce nucleic acids into cells, including forthe treatment of diseases.

SUMMARY OF INVENTION

The present invention provides novel cationic lipids, as well as lipidparticles comprising the same. These lipid particles may furthercomprise an active agent and be used according to related methods of theinvention to deliver the active agent to a cell.

The lipids of this invention may contain one or more isomeric forms. Allsuch isomeric forms of these compounds are expressly included in thepresent invention. The compounds of this invention may also containlinkages (e.g., carbon-carbon bonds) or substituents that can restrictbond rotation, e.g. restriction resulting from the presence of a doublebond. Accordingly, all cis/trans and E/Z isomers are expressly includedin the present invention.

In one aspect, the invention provides improved lipid formulationscomprising a cationic lipid of formula I, wherein formula I is:

Formula I can also be referred to as DLin-M-C3-DMA, MC3 or M-C3. Each ofFormula I, DLin-M-C3-DMA, MC3 and M-C3 have the formula as provideddirectly above.

Lipid formulations typically also comprise a neutral lipid, a sterol anda PEG or PEG-modified lipid.

In one aspect, the improved lipid formulation also includes a targetinglipid (e.g., a GalNAc and/or folate containing lipid).

In one aspect, the invention provides preparation for the improved lipidformulations via an extrusion or an in-line mixing method.

In one aspect, the invention further provides a method of administeringthe improved lipid formulations containing RNA-based construct to ananimal, and evaluating the expression of the target gene.

In one aspect, a lipid formulation featured in the invention, such as alipid formulation complexed with an oligonucleotide, such as a doublestranded RNA (dsRNA), can be used to modify (e.g., decrease) target geneexpression in a tumor cell in vivo or in vitro. In some embodiments, alipid formulation featured in the invention can be used to modify targetgene expression in a tumor cell line, including but not limited to HeLa,HCT116, A375, MCF7, B16F10, Hep3b, HUH7, HepG2, Skov3, U87, and PC3 celllines.

In another aspect, the invention provides a lipid particle comprisingthe lipid of the present invention. In certain embodiments, the lipidparticle further comprises a neutral lipid and a lipid capable ofreducing particle aggregation. In one embodiment, the lipid particleconsists essentially of (i) at least one lipid of the present invention;(ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii)sterol, e.g. cholesterol; and (iv) peg-lipid, e.g. PEG-DMG or PEG-cDMA,in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid. In one embodiment, the lipid ofthe present invention is optically pure.

In additional related embodiments, the present invention includes lipidparticles of the invention that further comprise therapeutic agent. Inone embodiment, the therapeutic agent is a nucleic acid. In oneembodiment, the nucleic acid is a plasmid, an immunostimulatoryoligonucleotide, a single stranded oligonucleotide, e.g. an antisenseoligonucleotide, an antagomir; a double stranded oligonucleotide, e.g. asiRNA; an aptamer or a ribozyme.

In yet another related embodiment, the present invention includes apharmaceutical composition comprising a lipid particle of the presentinvention and a pharmaceutically acceptable excipient, carrier ofdiluent.

The present invention further includes, in other related embodiments, amethod of modulating the expression of a target gene in a cell, themethod comprising providing to a cell a lipid particle or pharmaceuticalcomposition of the present invention. The target gene can be a wild typegene. In another embodiment, the target gene contains one or moremutations. In a particular embodiment, the method comprises specificallymodulating expression of a target gene containing one or more mutations.In particular embodiments, the lipid particle comprises a therapeuticagent selected from an immunostimulatory oligonucleotide, a singlestranded oligonucleotide, e.g. an antisense oligonucleotide, anantagomir, a double stranded oligonucleotide, e.g. a siRNA, an aptamer,a ribozyme. In one embodiment, the nucleic acid is plasmid that encodesa siRNA, an antisense oligonucleotide, an aptamer or a ribozyme.

In one aspect of the invention, the target gene is selected from thegroup consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV,PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene,MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene,JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene,Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene,PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene,topoisomerase I gene, topoisomerase II alpha gene, p73 gene,p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene, caveolin Igene, MIB I gene, MTAI gene, M68 gene, SORT1 gene, XBP1 gene, mutationsin tumor suppressor genes, p53 tumor suppressor gene, and combinationsthereof.

In another embodiment, the nucleic acid is a plasmid that encodes apolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In yet a further related embodiment, the present invention includes amethod of treating a disease or disorder characterized by overexpressionof a polypeptide in a subject, comprising providing to the subject alipid particle or pharmaceutical composition of the present invention,wherein the therapeutic agent is selected from an siRNA, a microRNA, anantisense oligonucleotide, and a plasmid capable of expressing an siRNA,a microRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In a further embodiment, the present invention includes a method ofinducing an immune response in a subject, comprising providing to thesubject a pharmaceutical composition of the present invention, whereinthe therapeutic agent is an immunostimulatory oligonucleotide. Inparticular embodiments, the pharmaceutical composition is provided tothe patient in combination with a vaccine or antigen.

In a related embodiment, the present invention includes a vaccinecomprising the lipid particle of the present invention and an antigenassociated with a disease or pathogen. In one embodiment, the lipidparticle comprises an immunostimulatory nucleic acid or oligonucleotide.In a particular embodiment, the antigen is a tumor antigen. In anotherembodiment, the antigen is a viral antigen, a bacterial antigen, or aparasitic antigen.

The present invention further includes methods of preparing the lipidparticles and pharmaceutical compositions of the present invention, aswell as kits useful in the preparation of these lipid particle andpharmaceutical compositions.

In another aspect, the invention provides a method of evaluating acomposition that includes an agent, e.g. a therapeutic agent ordiagnostic agent, and a lipid of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph depicting the effect of lipid formulationsincluding DLin-M-C3-DMA on the silencing of FVII in a mouse model.

FIG. 2 is a bar graph depicting the dose response of MC3 in rats withvarious liposomal compositions.

FIG. 3 is a bar graph that shows the ApoE dependence of efficacy offormulations comprising MC3. Wildtype but not ApoE knockout mice showeddose-dependent reduction in FVII protein levels. FIG. 2 also depicts agraph that demonstrates that ApoE dependence of the MC3 liposomalformulation and the lack of silencing in ApoE KO mice using MC3 can beeffectively rescued by premixing with ApoE.

FIG. 4 is a bar graph that shows the effects of variations in the molepercentage of MC3 in a liposomal formulation and also the effects ofvariations in the neutral lipid (e.g., varying the neutral lipid withDSPC, DMPC, and DLPC).

FIG. 5 is a bar graph showing that increasing PEG-shielding decreasesnon-GalNAc-mediated silencing in mice.

FIG. 6 is a bar graph showing that increasing PEG-shielding decreasesnon-GalNAc-mediated silencing in rats.

FIG. 7 is a bar graph showing the efficacy of liposomal formulationshaving different mol % of MC3, with and without GalNAc.

FIG. 8 is a bar graph showing that the activity of GalNAc-targetedliposomes is abolished in Asialoglycoprotein Receptor (ASGPR) knockoutmice.

FIG. 9 is a dose response curve of % residual FVII and dose (mg/kg) forthe formulation prepared in Example 17.

FIG. 10 is the pKa titration curve of a cationic lipid of formula I asdetermined in Example 18.

DETAILED DESCRIPTION

Described herein is an improved lipid formulation, which can be used,for example, as a delivering an agent, e.g., a nucleic acid-based agent,such as an RNA-based construct, to a cell or subject. Also describedherein are methods of administering the improved lipid formulationscontaining an RNA-based construct to an animal, and in some embodiments,evaluating the expression of the target gene. In some embodiments theimproved lipid formulation includes a targeting lipid (e.g., a targetinglipid described herein such as a GalNAc or folate containing lipid).

Lipids

The invention provides improved lipid formulations comprising a cationiclipid of formula I, a neutral lipid, a sterol and a PEG or PEG-modifiedlipid, wherein formula I is

In one embodiment, the lipid is a racemic mixture.

In one embodiment, the lipid is enriched in one diastereomer, e.g. thelipid has at least 95%, at least 90%, at least 80% or at least 70%diastereomeric excess.

In one embodiment, the lipid is chirally pure, e.g. is a single isomer.

In one embodiment, the lipid is enriched for one isomer.

In one embodiment, the formulations of the invention are entrapped by atleast 75%, at least 80% or at least 90%. In one embodiment, theformulation include from about 25% to about 75% on a molar basis ofcationic lipid of formula I e.g., from about 35 to about 65%, from about45 to about 65%, about 60%, about 57.5%, about 50% or about 40% on amolar basis.

In one embodiment, the formulation includes from about 0.5% to about 15%on a molar basis of the neutral lipid e.g., from about 3 to about 12%,from about 5 to about 10% or about 15%, about 10%, or about 7.5% on amolar basis.

In one embodiment, the formulation includes from about 5% to about 50%on a molar basis of the sterol (e.g., about 15 to about 45%, about 20 toabout 40%, about 40%, about 38.5%, about 35%, or about 31% on a molarbasis. In one embodiment, the sterol is cholesterol.

In one embodiment, the formulation includes from about 0.5% to about 20%on a molar basis of the PEG or PEG-modified lipid (e.g., about 0.5 toabout 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%,about 3.5%, or about 5% on a molar basis.

In one embodiment, the formulations of the inventions include 25-75% ofcationic lipid of formula I, 0.5-15% of the neutral lipid, 5-50% of thesterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include 35-65% ofcationic lipid of formula I, 3-12% of the neutral lipid, 15-45% of thesterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include 45-65% ofcationic lipid of formula I, 5-10% of the neutral lipid, 25-40% of thesterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about 60%of cationic lipid of formula I, about 7.5% of the neutral lipid, about31% of the sterol, and about 1.5% of the PEG or PEG-modified lipid on amolar basis. In one preferred embodiment, the cationic lipid is thecompound of formula I, the neutral lipid is DSPC, the sterol ischolesterol and the PEG lipid is PEG-DMG (also referred herein asPEG-C14 or C14-PEG). In one embodiment, the PEG or PEG modified lipidcomprises a PEG molecule of an average molecular weight of 2,000 Da. Inother embodiments, the PEG or PEG modified lipid comprises a PEGmolecule of an average molecular weight of less than 2,000, for examplearound 1,500 Da, around 1,000 Da, or around 500 Da. In one embodiment,the PEG or PEG modified lipid is a compound of the following Formula VI:

with a PEG molecule of an average molecular weight of 2,000 Da. In oneembodiment, the PEG or PEG modified lipid is PEG-distearoyl glycerol(PEG-DSG, also referred herein as PEG-C18 or C18-PEG).

In one embodiment, the formulations of the inventions include about 50%of cationic lipid of formula I, about 10% of the neutral lipid, about38.5% of the sterol, and about 1.5% of the PEG or PEG-modified lipid ona molar basis. In one preferred embodiment, the cationic lipid is thecompound of formula I, the neutral lipid is DSPC, the sterol ischolesterol and the PEG lipid is PEG-DMG (also referred herein asPEG-C14 or C14-PEG). In one embodiment, the PEG or PEG modified lipid isPEG-distyryl glycerol (PEG-DSG, also referred herein as PEG-C18 orC18-PEG). In one embodiment, the PEG or PEG modified lipid is PEG-DPG(PEG-dipalmitoylglycerol). In one embodiment, the PEG or PEG modifiedlipid comprises a PEG molecule of an average molecular weight of 2,000Da.

In one embodiment, the formulations of the inventions include about 50%of cationic lipid of formula I, about 10% of the neutral lipid, about35% of the sterol, about 4.5% of the PEG or PEG-modified lipid, andabout 0.5% of the targeting lipid on a molar basis. In one preferredembodiment, the cationic lipid is the compound of formula I, the neutrallipid is DSPC, the sterol is cholesterol, the PEG lipid isPEG-distearoyl glycerol (PEG-DSG, also referred herein as PEG-C18 orC18-PEG), and the targeting lipid is GalNAc3-PEG-DSG.

In one embodiment, the formulations of the inventions include about 50%of cationic lipid of formula I, about 10% of the neutral lipid, about35% of the sterol, about 4.5% of the PEG or PEG-modified lipid, andabout 0.5% of the targeting lipid on a molar basis. In one preferredembodiment, the cationic lipid is the compound of formula I, the neutrallipid is DSPC, the sterol is cholesterol, the PEG lipid is PEG-DMG (alsoreferred herein as PEG-C14 or C14-PEG).

In one embodiment, the formulations of the inventions include about 40%of cationic lipid of formula I, about 15% of the neutral lipid, about40% of the sterol, and about 5% of the PEG or PEG-modified lipid on amolar basis. In one preferred embodiment, the cationic lipid is thecompound of formula I, the neutral lipid is DSPC, the sterol ischolesterol, the PEG lipid is PEG-DMG (also referred herein as PEG-C14or C14-PEG).

In one embodiment, the formulations of the inventions include about 50%of cationic lipid of formula I, about 10% of the neutral lipid, about35% of the sterol, and about 5% of the PEG or PEG-modified lipid on amolar basis. In one preferred embodiment, the cationic lipid is thecompound of formula I, the neutral lipid is DSPC, the sterol ischolesterol, the PEG lipid is PEG-DMG (also referred herein as PEG-C14or C14-PEG).

In one embodiment, the formulations of the inventions include about57.2% of cationic lipid of formula I, about 7.1% of the neutral lipid,about 34.3% of the sterol, and about 1.4% of the PEG or PEG-modifiedlipid on a molar basis. In one preferred embodiment, the cationic lipidis the compound of formula I, the neutral lipid is DPPC, the sterol ischolesterol, the PEG lipid is PEG-cDMA (PEG-cDMA is further discussed inHeyes et al. (J. Controlled Release, 107, 276-287 (2005)).

GalNAc3-PEG-DSG In one embodiment, the PEG or PEG modified lipid is acompound of the Formula VI or PEG-DSG, wherein the PEG molecule has anaverage molecular weight of 2,000 Da.

In one embodiment, the formulations of the inventions include about57.5% of cationic lipid of formula I, about 7.5% of the neutral lipid,about 31.5% of the sterol, and about 3.5% of the PEG or PEG-modifiedlipid on a molar basis. In one preferred embodiment, the cationic lipidis the compound of formula I, the neutral lipid is DSPC, the sterol ischolesterol and the PEG lipid is PEG-DMG.

In one embodiment, the ratio of lipid:siRNA is at least about 0.5:1, atleast about 1:1, at least about 2:1, at least about 3:1, at least about4:1, at least about 5:1, at least about 6:1, at least about 7:1, atleast about 8:1, at least about 10:1, at least about 11:1, at leastabout 12:1, to at least about 15:1. In one embodiment, the ratio oflipid:siRNA ratio is between about 1:1 to about 20:1, about 3:1 to about15:1, about 4:1 to about 15:1, about 5:1 to about 13:1. In oneembodiment, the ratio of lipid:siRNA ratio is between about 0.5:1 toabout 15:1.

In one aspect, the improved lipid formulation also includes a targetinglipid. In some embodiments, the targeting lipid includes a GalNAc moiety(i.e., an N-galactosamine moiety). For example, a targeting lipidincluding a GalNAc moiety can include those disclosed in U.S. Ser. No.12/328,669, filed Dec. 4, 2008, which is incorporated herein byreference in its entirety. A targeting lipid can also include any otherlipid (e.g., targeting lipid) known in the art, for example, asdescribed in U.S. Ser. No. 12/328,669, or International Publication No.WO 2008/042973, the contents of each of which are incorporated herein byreference in their entirety. In some embodiments, the targeting lipidincludes a plurality of GalNAc moieties, e.g., two or three GalNAcmoieties. In some embodiments, the targeting lipid contains a plurality,e.g., two or three N-acetylgalactosamine (GalNAc) moieties. In someembodiments, the lipid in the targeting lipid is1,2-Di-O-hexadecyl-sn-glyceride (i.e., DSG). In some embodiments, thetargeting lipid includes a PEG moiety (e.g., a PEG moiety having amolecular weight of at least about 500 Da, such as about 1000 Da, 1500Da, 2000 Da or greater), for example, the targeting moiety is connectedto the lipid via a PEG moiety.

In some embodiments, the targeting lipid includes a folate moiety. Forexample, a targeting lipid including a folate moiety can include thosedisclosed in U.S. Ser. No. 12/328,669, filed Dec. 4, 2008, which isincorporated herein by reference in its entirety. In another embodiment,a targeting lipid including a folate moiety can include the compound ofFormula V.

Exemplary targeting lipids are represented by formula L below:(Targeting group)_(n)-L-Lipid   formula L

wherein

Targeting group is any targeting group that known by one skilled in theart and/or described herein (e.g., a cell surface receptor);

n is an integer from 1 to 5, (e.g., 3)

L is a linking group; and

Lipid is a lipid such as a lipid described herein (e.g., a neutral lipidsuch as DSG).

In some embodiments, the linking group includes a PEG moiety.

In some embodiments, the targeting lipid is compound II, III, IV or V asprovided below:

In some embodiments, the targeting lipid is present in the formulationin an amount of from about 0.001% to about 5% (e.g., about 0.005%,0.15%, 0.3%, 0.5%, 1.5%, 2%, 2.5%, 3%, 4%, or 5%) on a molar basis. Insome embodiments, the targeting lipid is present in the formulation inan amount from about 0.005% to about 1.5%. In some embodiments, thetargeting lipid is included in a formulation described herein.

In some embodiments, the lipid formulation also included an antioxidant(e.g., a radical scavenger). The antioxidant can be present in theformulation, for example, at an around from about 0.01% to about 5%. Theantioxidant can be hydrophobic or hydrophilic (e.g., soluble in lipidsor soluble in water). In some embodiments, the antioxidant is a phenoliccompound, for example, butylhydroxytoluene, resveratrol, coenzyme Q10,or other flavinoids, or a vitamin, for example, vitamin E or vitamin C.Other exemplary antioxidants include lipoic acid, uric acid, a carotenesuch as beta-carotene or retinol (vitamin A), glutathione, melatonin,selenium, and ubiquinol.

In some embodiments, the receptor for the targeting lipid (e.g., aGalNAc containing lipid) is the asialoglycoprotein receptor (i.e.,ASGPR).

In one embodiment, the formulations of the invention are produced via anextrusion method or an in-line mixing method.

The extrusion method (also refer to as preformed method or batchprocess) is a method where the empty liposomes (i.e. no nucleic acid)are prepared first, followed by the addition of nucleic acid to theempty liposome. Extrusion of liposome compositions through a small-porepolycarbonate membrane or an asymmetric ceramic membrane results in arelatively well-defined size distribution. Typically, the suspension iscycled through the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing. Thesemethods are disclosed in the U.S. Pat. No. 5,008,050; U.S. Pat. No.4,927,637; U.S. Pat. No. 4,737,323; Biochim Biophys Acta. 1979 Oct. 19;557(1):9-23; Biochim Biophys Acta. 1980 Oct. 2; 601(3):559-7; BiochimBiophys Acta. 1986 Jun. 13; 858(1):161-8; and Biochim. Biophys. Acta1985 812, 55-65, which are hereby incorporated by reference in theirentirety.

The in-line mixing method is a method wherein both the lipids and thenucleic acid are added in parallel into a mixing chamber. The mixingchamber can be a simple T-connector or any other mixing chamber that isknown to one skill in the art. These methods are disclosed in U.S. Pat.No. 6,534,018 and U.S. Pat. No. 6,855,277; US publication 2007/0042031and Pharmaceuticals Research, Vol. 22, No. 3, March 2005, p. 362-372,which are hereby incorporated by reference in their entirety.

It is further understood that the formulations of the invention can beprepared by any methods known to one of ordinary skill in the art.

In a further embodiment, representative formulations comprising thecompound of formula I, are delineated in Table 1.

TABLE 1 MC3 DSPC Cholesterol PEG 60   7.5 31 1.5 50 10 38.5 1.5 40 2038.5 1.5 50 10 38.5 1.5 50 10 38.5 1.5 40 20 38.5 1.5 60   7.5 21 1.5 5010 38.5 1.5 50 10 38.5 1.5 40 20 38.5 1.5 (DMPC) 30 30 38.5 1.5 50 1038.5 1.5 (DMPC) 30 30 38.5 1.5 (DMPC) 51 10 38.5 1.5 (DLPC) 40 20 38.51.5 (DLPC) 40 20 38.5 1.5 40 10 40 10 60 10 20 10 40 20 37 3 60 10 27 3

In one embodiment, specific formulations comprising the compound offormula I are described as follows:

Ratio of Lipids (in Molar Percentage)

Lipid:siRNA Ratio

50/10/38.5/1.5 (MC3:DSPC:Cholesterol:PEG-DMG)

Lipid:siRNA ˜11

40/15/40/5 (MC3:DSPC:Cholesterol:PEG-DMG)

Lipid:siRNA ratio ˜11

50/10/35/4.5/0.5% (MC3:DSPC:Cholesterol:PEG-DSG (C18-PEG):GalNAc3-PEG-DSG)

Lipid:siRNA ratio ˜11

50/10/30/9.5/0.5% (MC3:DSPC:Cholesterol:PEG-DSG:GalNAc3-PEG-DSG)

Lipid:siRNA ratio ˜11

50/10/35/5% (MC3:DSPC:Cholesterol:PEG-DSG

Lipid:siRNA ratio ˜11

50/10/38.5/1.5 (MC3:DPPC:Cholesterol:PEG-DMG)

Lipid:siRNA ˜11

40/15/40/5 (MC3:DPPC:Cholesterol:PEG-DMG)

Lipid:siRNA ratio ˜11

50/10/35/4.5/0.5%. (MC3:DPPC:Cholesterol:PEG-DSG:GalNAc3-PEG-DSG)

Lipid:siRNA ratio ˜11

50/10/30/9.5/0.5% (MC3:DPPC:Cholesterol:PEG-DSG:GalNAc3-PEG-DSG)

Lipid:siRNA ratio ˜11

50/10/35/5% (MC3:DPPC:Cholesterol:PEG-DSG

Lipid:siRNA ratio ˜11

50/10/38.5/1.5 (MC3:DSPC:Cholesterol:PEG-DMG)

Lipid:siRNA ˜7

50/10/38.5/1.5 (MC3:DSPC:Cholesterol:PEG-DSG)

Lipid:siRNA ˜10

50/10/38.5/1.5 (MC3:DSPC:Cholesterol:PEG-DMG)

Lipid:siRNA ˜12

50/10/35/5% (MC3:DSPC:Cholesterol:PEG-DMG

Lipid:siRNA ratio ˜8

50/10/35/5% (MC3:DSPC:Cholesterol:PEG-DMG

Lipid:siRNA ratio ˜10

In one embodiment, the formulations of the invention are entrapped by atleast 75%, at least 80% or at least 90%.

In one embodiment, the formulations of the invention further comprise anapolipoprotein. As used herein, the term “apolipoprotein” or“lipoprotein” refers to apolipoproteins known to those of skill in theart and variants and fragments thereof and to apolipoprotein agonists,analogues or fragments thereof described below.

Suitable apolipoproteins include, but are not limited to, ApoA-I,ApoA-II, ApoA-IV, ApoA-V and ApoE, and active polymorphic forms,isoforms, variants and mutants as well as fragments or truncated formsthereof. In certain embodiments, the apolipoprotein is a thiolcontaining apolipoprotein. “Thiol containing apolipoprotein” refers toan apolipoprotein, variant, fragment or isoform that contains at leastone cysteine residue. The most common thiol containing apolipoproteinsare ApoA-I Milano (ApoA-I_(M)) and ApoA-I Paris (ApoA-I_(P)) whichcontain one cysteine residue (Jia et al., 2002, Biochem. Biophys. Res.Comm. 297: 206-13; Bielicki and Oda, 2002, Biochemistry 41: 2089-96).ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins.Isolated ApoE and/or active fragments and polypeptide analogues thereof,including recombinantly produced forms thereof, are described in U.S.Pat. Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189;5,168,045; 5,116,739; the disclosures of which are herein incorporatedby reference. ApoE3 is disclosed in Weisgraber, et al., “Human Eapoprotein heterogeneity: cysteine-arginine interchanges in the aminoacid sequence of the apo-E isoforms,” J. Biol. Chem. (1981) 256:9077-9083; and Rall, et al., “Structural basis for receptor bindingheterogeneity of apolipoprotein E from type III hyperlipoproteinemicsubjects,” Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. See also GenBankaccession number K00396.

In certain embodiments, the apolipoprotein can be in its mature form, inits preproapolipoprotein form or in its proapolipoprotein form. Homo-and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger etal., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-IMilano (Klon et al., 2000, Biophys. J. 79:(3)1679-87; Franceschini etal., 1985, J. Biol. Chem. 260: 1632-35), ApoA-1 Paris (Dawn et al.,1999, J. Mol. Med. 77:614-22), ApoA-H (Shelness et al., 1985, J. Biol.Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.258(14):8993-9000) can also be utilized within the scope of theinvention.

In certain embodiments, the apolipoprotein can be a fragment, variant orisoform of the apolipoprotein. The term “fragment” refers to anyapolipoprotein having an amino acid sequence shorter than that of anative apolipoprotein and which fragment retains the activity of nativeapolipoprotein, including lipid binding properties. By “variant” ismeant substitutions or alterations in the amino acid sequences of theapolipoprotein, which substitutions or alterations, e.g., additions anddeletions of amino acid residues, do not abolish the activity of nativeapolipoprotein, including lipid binding properties. Thus, a variant cancomprise a protein or peptide having a substantially identical aminoacid sequence to a native apolipoprotein provided herein in which one ormore amino acid residues have been conservatively substituted withchemically similar amino acids. Examples of conservative substitutionsinclude the substitution of at least one hydrophobic residue such asisoleucine, valine, leucine or methionine for another. Likewise, thepresent invention contemplates, for example, the substitution of atleast one hydrophilic residue such as, for example, between arginine andlysine, between glutamine and asparagine, and between glycine and serine(see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term“isoform” refers to a protein having the same, greater or partialfunction and similar, identical or partial sequence, and may or may notbe the product of the same gene and usually tissue specific (seeWeisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J.Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem.260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon etal., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol.18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90;Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov etal., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985,J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem.255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sacre etal., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003, Biophys.Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.277(33):29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23):14888-93 andU.S. Pat. No. 6,372,886).

In certain embodiments, the methods and compositions of the presentinvention include the use of a chimeric construction of anapolipoprotein. For example, a chimeric construction of anapolipoprotein can be comprised of an apolipoprotein domain with highlipid binding capacity associated with an apolipoprotein domaincontaining ischemia reperfusion protective properties. A chimericconstruction of an apolipoprotein can be a construction that includesseparate regions within an apolipoprotein (i.e., homologousconstruction) or a chimeric construction can be a construction thatincludes separate regions between different apolipoproteins (i.e.,heterologous constructions). Compositions comprising a chimericconstruction can also include segments that are apolipoprotein variantsor segments designed to have a specific character (e.g., lipid binding,receptor binding, enzymatic, enzyme activating, antioxidant orreduction-oxidation property) (see Weisgraber 1990, J. Lipid Res.31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35;Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al, 1986, J.Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem.259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al.,1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al.,1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, DrugMetab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem.275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71;Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sorenson et al., 1999,Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996,Arterioscler. Throb. Vasc. Biol. 16(2):328-38: Thurberg et al., J. Biol.Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill1998, J. Biol. Chem. 273(47):30979-84).

Apolipoproteins utilized in the invention also include recombinant,synthetic, semi-synthetic or purified apolipoproteins. Methods forobtaining apolipoproteins or equivalents thereof, utilized by theinvention are well-known in the art. For example, apolipoproteins can beseparated from plasma or natural products by, for example, densitygradient centrifugation or immunoaffinity chromatography, or producedsynthetically, semi-synthetically or using recombinant DNA techniquesknown to those of the art (see, e.g., Mulugeta et al., 1998, J.Chromatogr. 798(1-2): 83-90; Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson,et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528,5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 andWO 87/02062).

Apolipoproteins utilized in the invention further include apolipoproteinagonists such as peptides and peptide analogues that mimic the activityof ApoA-I, ApoA-I Milano (ApoA-I_(M)), ApoA-I Paris (ApoA-I_(P)),ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be anyof those described in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166,and 5,840,688, the contents of which are incorporated herein byreference in their entireties.

Apolipoprotein agonist peptides or peptide analogues can be synthesizedor manufactured using any technique for peptide synthesis known in theart including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166. For example, the peptides may beprepared using the solid-phase synthetic technique initially describedby Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptidesynthesis techniques may be found in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques can be found in Stuart and Young, Solid PhasePeptide. Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).Peptides may also be synthesized by solution methods as described in TheProteins, Vol. II, 3d Ed., Neurath et. al., Eds., p. 105-237, AcademicPress, New York, N.Y. (1976). Appropriate protective groups for use indifferent peptide syntheses are described in the above-mentioned textsas well as in McOmie, Protective Groups in Organic Chemistry, PlenumPress, New York, N.Y. (1973). The peptides of the present inventionmight also be prepared by chemical or enzymatic cleavage from largerportions of, for example, apolipoprotein A-I.

In certain embodiments, the apolipoprotein can be a mixture ofapolipoproteins. In one embodiment, the apolipoprotein can be ahomogeneous mixture, that is, a single type of apolipoprotein. Inanother embodiment, the apolipoprotein can be a heterogeneous mixture ofapolipoproteins, that is, a mixture of two or more differentapolipoproteins. Embodiments of heterogenous mixtures of apolipoproteinscan comprise, for example, a mixture of an apolipoprotein from an animalsource and an apolipoprotein from a semi-synthetic source. In certainembodiments, a heterogenous mixture can comprise, for example, a mixtureof ApoA-I and ApoA-I Milano. In certain embodiments, a heterogeneousmixture can comprise, for example, a mixture of ApoA-I Milano and ApoA-IParis. Suitable mixtures for use in the methods and compositions of theinvention will be apparent to one of skill in the art.

If the apolipoprotein is obtained from natural sources, it can beobtained from a plant or animal source. If the apolipoprotein isobtained from an animal source, the apolipoprotein can be from anyspecies. In certain embodiments, the apolipoprotein can be obtained froman animal source. In certain embodiments, the apolipoprotein can beobtained from a human source. In preferred embodiments of the invention,the apolipoprotein is derived from the same species as the individual towhich the apolipoprotein is administered.

In one embodiment, the target gene is selected from the group consistingof Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene,Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNKgene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOSgene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene,Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKBgene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene,topoisomerase II alpha gene, p73 gene, p21(WAF1/CIP1) gene, p27(KIP1)gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68gene, mutations in tumor suppressor genes, p53 tumor suppressor gene,and combinations thereof. In one embodiment the target gene is a geneexpressed in the liver, e.g., the Factor VII (FVII) gene. The effect ofthe expression of the target gene, e.g., FVII, is evaluated by measuringFVII levels in a biological sample, such as a serum or tissue sample.For example, the level of FVII, e.g., as measured by assay of FVIIactivity, in blood can be determined. In one embodiment, the level ofmRNA in the liver can be evaluated. In another preferred embodiment, atleast two types of evaluation are made, e.g., an evaluation of proteinlevel (e.g., in blood), and a measure of mRNA level (e.g., in the liver)are both made.

In one embodiment, the agent is a nucleic acid, such as adouble-stranded RNA (dsRNA).

In another embodiment, the nucleic acid agent is a single-stranded DNAor RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid. For example, adouble-stranded DNA can be a structural gene, a gene including controland termination regions, or a self-replicating system such as a viral orplasmid DNA. A double-stranded RNA can be, e.g., a dsRNA or another RNAinterference reagent. A single-stranded nucleic acid can be, e.g., anantisense oligonucleotide, ribozyme, microRNA, or triplex-formingoligonucleotide.

In yet another embodiment, at various time points after administrationof a candidate agent, a biological sample, such as a fluid sample, e.g.,blood, plasma, or serum, or a tissue sample, such as a liver sample, istaken from the test subject and tested for an effect of the agent ontarget protein or mRNA expression levels. In one particularly preferredembodiment, the candidate agent is a dsRNA that targets FVII, and thebiological sample is tested for an effect on Factor VII protein or mRNAlevels. In one embodiment, plasma levels of FVII protein are assayed,such as by using an immunohistochemistry assay or a chromogenic assay.In another embodiment, levels of FVII mRNA in the liver are tested by anassay, such as a branched DNA assay, or a Northern blot or RT-PCR assay.

In one embodiment, the agent, e.g., a composition including the improvedlipid formulation, is evaluated for toxicity. In yet another embodiment,the model subject can be monitored for physical effects, such as by achange in weight or cageside behavior.

In one embodiment, the method further includes subjecting the agent,e.g., a composition comprising the improved lipid formulation, to afurther evaluation. The further evaluation can include, for example, (i)a repetition of the evaluation described above, (ii) a repetition of theevaluation described above with a different number of animals or withdifferent doses, or (iii) by a different method, e.g., evaluation inanother animal model, e.g., a non-human primate.

In another embodiment, a decision is made regarding whether or not toinclude the agent and the improved lipid formulation in further studies,such as in a clinical trial, depending on the observed, effect of thecandidate agent on liver protein or mRNA levels. For example, if acandidate dsRNA is observed to decrease protein or mRNA levels by atleast 20%, 30%, 40%, 50%, or more, then the agent can be considered fora clinical trial.

In yet another embodiment, a decision is made regarding whether or notto include the agent and the improved lipid formulation in apharmaceutical composition, depending on the observed effect of thecandidate agent and amino lipid on liver protein or mRNA levels. Forexample, if a candidate dsRNA is observed to decrease protein or mRNAlevels by at least 20%, 30%, 40%, 50%, or more, then the agent can beconsidered for a clinical trial.

In another aspect, the invention features a method of evaluating theimproved lipid formulation for its suitability for delivering atherapeutic agent to a cell. In some embodiments, the invention featuresa method of evaluating the improved lipid formulation for itssuitability for delivering an RNA-based construct, e.g., a dsRNA thattargets FVII. The method includes providing a composition that includesa dsRNA that targets FVII and a candidate amino lipid, administering thecomposition to a rodent, e.g., a mouse, evaluating the expression ofFVII as a function of at least one of the level of FVII in the blood orthe level of FVII mRNA in the liver, thereby evaluating the candidateamino lipid. In some embodiments, the method further comprises comparingexpression of the target gene with a preselected reference value.

Compositions that include lipid containing components, such as aliposome, and these are described in greater detail below. Exemplarynucleic acid-based agents include dsRNAs, antisense oligonucleotides,ribozymes, microRNAs, immunostimulatory oligonucleotides, ortriplex-forming oligonucleotides. These agents are also described ingreater detail below.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizinyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x),—C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y),wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different andindependently hydrogen, alkyl or heterocycle, and each of said alkyl andheterocycle substituents may be further substituted with one or more ofoxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), ═C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond. The term“alkylheterocycle” refers to a heteroaryl wherein at least one of thering atoms is substituted with alkyl, alkenyl or alkynyl

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio,alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl,arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl,haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino,alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl,carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl,aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonicacid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understoodthat the substituent may be further substituted. “Halogen” means fluoro,chloro, bromo and iodo.

The terms “alkylamine” and “dialkylamine” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively.

The term “alkylphosphate” refers to —O—P(Q′)(Q″)-O—R, wherein Q′ and Q″are each independently O, S, N(R)₂, optionally substituted alkyl oralkoxy; and R is optionally substituted alkyl, ω-aminoalkyl orω-(substituted)aminoalkyl.

The term “alkylphosphorothioate” refers to an alkylphosphate wherein atleast one of Q′ or Q″ is S.

The term “alkylphosphonate” refers to an alkylphosphate wherein at leastone of Q′ or Q″ is alkyl.

The term “hydroxyalkyl” means —O-alkyl radical.

The term “alkylheterocycle” refers to an alkyl where at least onemethylene has been replaced by a heterocycle.

The term “ω-aminoalkyl” refers to -alkyl-NH₂ radical. And the term“ω-(substituted)aminoalkyl refers to an ω-aminoalkyl wherein at leastone of the H on N has been replaced with alkyl.

The term “ω-phosphoalkyl” refers to -alkyl-O—P(Q′)(Q″)-O—R, wherein Q′and Q″ are each independently O or S and R optionally substituted alkyl.

The term “ω-thiophosphoalkyl refers to ω-phosphoalkyl wherein at leastone of Q′ or Q″ is S.

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, Green, T. W. et. al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Lipid Particles

The agents and/or amino lipids for testing in the liver screening modelfeatured herein can be formulated in lipid particles. Lipid particlesinclude, but are not limited to, liposomes. As used herein, a liposomeis a structure having lipid-containing membranes enclosing an aqueousinterior. Liposomes may have one or more lipid membranes. The inventioncontemplates both single-layered liposomes, which are referred to asunilamellar, and multi-layered liposomes, which are referred to asmultilamellar. When complexed with nucleic acids, lipid particles mayalso be lipoplexes, which are composed of cationic lipid bilayerssandwiched between DNA layers, as described, e.g., in Felgner,Scientific American.

Lipid particles may further include one or more additional lipids and/orother components such as cholesterol. Other lipids may be included inthe liposome compositions for a variety of purposes, such as to preventlipid oxidation or to attach ligands onto the liposome surface. Any of anumber of lipids may be present, including amphipathic, neutral,cationic, and anionic lipids. Such lipids can be used alone or incombination. Specific examples of additional lipid components that maybe present are described below.

Additional components that may be present in a lipid particle includebilayer stabilizing components such as polyamide oligomers (see, e.g.,U.S. Pat. No. 6,320,017), peptides, proteins, detergents,lipid-derivatives, such as PEG coupled to phosphatidylethanolamine andPEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613). In someembodiments, the lipid particle includes a targeting agent such as atargeting lipid described herein.

A lipid particle can include one or more of a second amino lipid orcationic lipid, a neutral lipid, a sterol, and a lipid selected toreduce aggregation of lipid particles during formation, which may resultfrom steric stabilization of particles which prevents charge-inducedaggregation during formation.

As used herein, the term “cationic lipid” is meant to include thoselipids having one or two fatty acid or fatty alkyl chains and an aminohead group (including an alkylamino or dialkylamino group) that may beprotonated to form a cationic lipid at physiological pH. In someembodiments, a cationic lipid is referred to as an “amino lipid.”

Other cationic lipids would include those having alternative fatty acidgroups and other dialkylamino groups, including those in which the alkylsubstituents are different (e.g., N-ethyl-N-methylamino-,N-propyl-N-ethylamino- and the like). In general, lipids (e.g., acationic lipid) having less saturated acyl chains are more easily sized,particularly when the complexes are sized below about 0.3 microns, forpurposes of filter sterilization. Cationic lipids containing unsaturatedfatty acids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Other scaffolds can also be used to separate the amino group(e.g., the amino group of the cationic lipid) and the fatty acid orfatty alkyl portion of the cationic lipid. Suitable scaffolds are knownto those of skill in the art.

In certain embodiments, cationic lipids have at least one protonatableor deprotonatable group, such that the lipid is positively charged at apH at or below physiological pH (e.g. pH 7.4), and neutral at a secondpH, preferably at or above physiological pH. Such lipids are alsoreferred to as cationic lipids. It will, of course, be understood thatthe addition or removal of protons as a function of pH is an equilibriumprocess, and that the reference to a charged or a neutral lipid refersto the nature of the predominant species and does not require that allof the lipid be present in the charged or neutral form. Lipids that havemore than one protonatable or deprotonatable group, or which arezwiterrionic, are not excluded from use in the invention.

In certain embodiments, protonatable lipids (i.e., cationic lipids) havea pKa of the protonatable group in the range of about 4 to about 11.Most preferred is pKa of about 4 to about 7, because these lipids willbe cationic at a lower pH formulation stage, while particles will belargely (though not completely) surface neutralized at physiological pHaround pH 7.4. One of the benefits of this pKa is that at least somenucleic acid associated with the outside surface of the particle willlose its electrostatic interaction at physiological pH and be removed bysimple dialysis; thus greatly reducing the particle's susceptibility toclearance.

Examples of lipids that reduce aggregation of particles during formationinclude polyethylene glycol (PEG)-modified lipids, monosialogangliosideGm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No.6,320,017). Other compounds with uncharged, hydrophilic, steric-barriermoieties, which prevent aggregation during formulation, like PEG, Gm1 orATTA, can also be coupled to lipids for use as in the methods andcompositions of the invention. ATTA-lipids are described, e.g., in U.S.Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., inU.S. Pat. Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, theconcentration of the lipid component selected to reduce aggregation isabout 1 to 15% (by mole percent of lipids).

Examples of lipids that reduce aggregation and/or are suitable forconjugation to nucleic acid agents that can be used in the liverscreening model are polyethylene glycol (PEG)-modified lipids,monosialoganglioside Gm1, and polyamide oligomers (“PAO”) such as(described in U.S. Pat. No. 6,320,017). Other compounds with uncharged,hydrophilic, steric-barrier moieties, which prevent aggregation duringformulation, like PEG, Gm1 or ATTA, can also be coupled to lipids foruse as in the methods and compositions of the invention. ATTA-lipids aredescribed, e.g., in U.S. Pat. No. 6,320,017, and PEG-lipid conjugatesare described, e.g., in U.S. Pat. Nos. 5,820,873, 5,534,499 and5,885,613. Typically, the concentration of the lipid component selectedto reduce aggregation is about 1 to 15% (by mole percent of lipids).

Specific examples of PEG-modified lipids (or lipid-polyoxyethyleneconjugates) that are useful in the invention can have a variety of“anchoring” lipid portions to secure the PEG portion to the surface ofthe lipid vesicle. Examples of suitable PEG-modified lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which aredescribed in co-pending U.S. Ser. No. 08/486,214, incorporated herein byreference, PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modifieddiacylglycerols and dialkylglycerols. In some embodiments, the total mol% of PEG lipids within a particle is about 1.5 mol %. For example, whenthe particle includes a plurality of PEG lipids described herein such asa PEG-modified lipid as described above and a targeting lipid containinga PEG, the total amount of the PEG containing lipids when taken togetheris about 1.5 mol %.

In embodiments where a sterically-large moiety such as PEG or ATTA areconjugated to a lipid anchor, the selection of the lipid anchor dependson what type of association the conjugate is to have with the lipidparticle. It is well known that mePEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remainassociated with a liposome until the particle is cleared from thecirculation, possibly a matter of days. Other conjugates, such asPEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidlyexchanges out of the formulation upon exposure to serum, with a T_(1/2)less than 60 mins. in some assays. As illustrated in U.S. patentapplication Ser. No. 08/486,214, at least three characteristicsinfluence the rate of exchange: length of acyl chain, saturation of acylchain, and size of the steric-barrier head group. Compounds havingsuitable variations of these features may be useful for the invention.For some therapeutic applications it may be preferable for thePEG-modified lipid to be rapidly lost from the nucleic acid-lipidparticle in vivo and hence the PEG-modified lipid will possessrelatively short lipid anchors. In other therapeutic applications it maybe preferable for the nucleic acid-lipid particle to exhibit a longerplasma circulation lifetime and hence the PEG-modified lipid willpossess relatively longer lipid anchors. Exemplary lipid anchors includethose having lengths of from about C14 to about C₂₂, preferably fromabout C₁₄ to about C₁₆. In some embodiments, a PEG moiety, for examplean mPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or20,000 daltons.

It should be noted that aggregation preventing compounds do notnecessarily require lipid conjugation to function properly. Free PEG orfree ATTA in solution may be sufficient to prevent aggregation. If theparticles are stable after formulation, the PEG or ATTA can be dialyzedaway before administration to a subject.

Neutral lipids, when present in the lipid particle, can be any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Theselection of neutral lipids for use in the particles described herein isgenerally guided by consideration of, e.g., liposome size and stabilityof the liposomes in the bloodstream. Preferably, the neutral lipidcomponent is a lipid having two acyl groups, (i.e.,diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipidshaving a variety of acyl chain groups of varying chain length and degreeof saturation are available or may be isolated or synthesized bywell-known techniques. In one group of embodiments, lipids containingsaturated fatty acids with carbon chain lengths in the range of C₁₄ toC₂₂ are preferred. In another group of embodiments, lipids with mono ordiunsaturated fatty acids with carbon chain lengths in the range of C₁₄to C₂₂ are used. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. Preferably, the neutrallipids used in the invention are DOPE, DSPC, DPPC, POPC, or any relatedphosphatidylcholine. The neutral lipids useful in the invention may alsobe composed of sphingomyelin, dihydrosphingomyeline, or phospholipidswith other head groups, such as serine and inositol.

The sterol component of the lipid mixture, when present, can be any ofthose sterols conventionally used in the field of liposome, lipidvesicle or lipid particle preparation. A preferred sterol ischolesterol.

Other cationic lipids, which carry a net positive charge at aboutphysiological pH, in addition to those specifically described above, mayalso be included in lipid particles of the invention. Such cationiclipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammoniumchloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammoniumchloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL). In particular embodiments, acationic lipid is an amino lipid.

Anionic lipids suitable for use in lipid particles of the inventioninclude, but are not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, andother anionic modifying groups joined to neutral lipids.

In numerous embodiments, amphipathic lipids are included in lipidparticles of the invention. “Amphipathic lipids” refer to any suitablematerial, wherein the hydrophobic portion of the lipid material orientsinto a hydrophobic phase, while the hydrophilic portion orients towardthe aqueous phase. Such compounds include, but are not limited to,phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine(DOPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or dilinoleylphosphatidylcholine (DLPC). Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and β-acyloxy acids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols.

Also suitable for inclusion in the lipid particles of the invention areprogrammable fusion lipids. Such lipid particles have little tendency tofuse with cell membranes and deliver their payload until a given signalevent occurs. This allows the lipid particle to distribute more evenlyafter injection into an organism or disease site before it starts fusingwith cells. The signal event can be, for example, a change in pH,temperature, ionic environment, or time. In the latter case, a fusiondelaying or “cloaking” component, such as an ATTA-lipid conjugate or aPEG-lipid conjugate, can simply exchange out of the lipid particlemembrane over time. Exemplary lipid anchors include those having lengthsof from about C₁₄ to about C₂₂, preferably from about C₁₄ to about C₁₆.In some embodiments, a PEG moiety, for example an mPEG-NH₂, has a sizeof about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.

By the time the lipid particle is suitably distributed in the body, ithas lost sufficient cloaking agent so as to be fusogenic. With othersignal events, it is desirable to choose a signal that is associatedwith the disease site or target cell, such as increased temperature at asite of inflammation.

A lipid particle conjugated to a nucleic acid agent can also include atargeting moiety, e.g., a targeting moiety that is specific to a celltype or tissue. Targeting of lipid particles using a variety oftargeting moieties, such as ligands, cell surface receptors,glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies,has been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044). Exemplary targeting moieties include a targeting lipid suchas a targeting lipid described herein. In some embodiments, thetargeting lipid is a GalNAc containing targeting lipid such asGalNAc3-DSG and GalNAc3-PEG-DSG as described herein. The targetingmoieties can include the entire protein or fragments thereof. Targetingmechanisms generally require that the targeting agents be positioned onthe surface of the lipid particle in such a manner that the targetingmoiety is available for interaction with the target, for example, a cellsurface receptor. A variety of different targeting agents and methodsare known and available in the art, including those described, e.g., inSapra, P. and Allen, T M, Prog. Lipid Res. 42(5):439-62 (2003); andAbra, R M et al., J. Liposome Res. 12:1-3, (2002).

The use of lipid particles, i.e., liposomes, with a surface coating ofhydrophilic polymer chains, such as polyethylene glycol (PEG) chains,for targeting has been proposed (Allen, et al., Biochimica et BiophysicaActa 1237: 99-108 (1995); DeFrees, et al., Journal of the AmericanChemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica etBiophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal ofLiposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic andMartin, Eds) CRC Press, Boca Raton Fla. (1995). In one approach, aligand, such as an antibody, for targeting the lipid particle is linkedto the polar head group of lipids forming the lipid particle. In anotherapproach, the targeting ligand is attached to the distal ends of the PEGchains forming the hydrophilic polymer coating (Klibanov, et al.,Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBSLetters 388: 115-118 (1996)).

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.Examples of targeting moieties can also include other proteins, specificto cellular components, including antigens associated with neoplasms ortumors. Proteins used as targeting moieties can be attached to theliposomes via covalent bonds (see, Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

In one exemplary embodiment, the lipid particle comprises a mixture of acationic lipid of the present invention, neutral lipids (other than acationic lipid), a sterol (e.g., cholesterol) and a PEG-modified lipid(e.g., a PEG-DMG or PEG-cDMA). In certain embodiments, the lipid mixtureconsists of or consists essentially of a cationic lipid of the presentinvention, a neutral lipid, cholesterol, and a PEG-modified lipid. Infurther preferred embodiments, the lipid particle consists of orconsists essentially of the above lipid mixture in molar ratios of about20-70% DLin-M-C3-DMA:5-45% neutral lipid:20-55% cholesterol:0.5-15%PEG-modified lipid.

In particular embodiments, the lipid particle consists of or consistsessentially of DLin-M-C3-DMA, DSPC, Chol, and either PEG-DMG orPEG-cDMA, e.g., in a molar ratio of about 20-60% DLin-M-C3-DMA:5-25%DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-cDMA. In particular embodiments,the molar lipid ratio is approximately 40/10/40/10 (mol %DLin-M-C3-DMA/DSPC/Chol/PEG-DMG or PEG-cDMA), 35/15/40/10 (mol %DLin-M-C3-DMA/DSPC/Chol/PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol %DLin-M-C3-DMA/DSPC/Chol/PEG-DMG or PEG-cDMA).

In another group of embodiments, the neutral lipid, DSPC, in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

Therapeutic Agent-Lipid Particle Compositions and Formulations

The invention includes compositions comprising a lipid particle of theinvention and an active agent, wherein the active agent is associatedwith the lipid particle. In particular embodiments, the active agent isa therapeutic agent. In particular embodiments, the active agent isencapsulated within an aqueous interior of the lipid particle. In otherembodiments, the active agent is present within one or more lipid layersof the lipid particle. In other embodiments, the active agent is boundto the exterior or interior lipid surface of a lipid particle.

“Fully encapsulated” as used herein indicates that the nucleic acid inthe particles is not significantly degraded after exposure to serum or anuclease assay that would significantly degrade free DNA. In a fullyencapsulated system, preferably less than 25% of particle nucleic acidis degraded in a treatment that would normally degrade 100% of freenucleic acid, more preferably less than 10% and most preferably lessthan 5% of the particle nucleic acid is degraded. Alternatively, fullencapsulation may be determined by an Oligreen® assay. Oligreen® is anultra-sensitive fluorescent nucleic acid stain for quantitatingoligonucleotides and single-stranded DNA in solution (available fromInvitrogen Corporation, Carlsbad, Calif.). Fully encapsulated alsosuggests that the particles are serum stable, that is, that they do notrapidly decompose into their component parts upon in vivoadministration.

Active agents, as used herein, include any molecule or compound capableof exerting a desired effect on a cell, tissue, organ, or subject. Sucheffects may be biological, physiological, or cosmetic, for example.Active agents may be any type of molecule or compound, including e.g.,nucleic acids, peptides and polypeptides, including, e.g., antibodies,such as, e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, and Primatized™ antibodies, cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell surfacereceptors and their ligands; hormones; and small molecules, includingsmall organic molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt orderivative thereof. Therapeutic agent derivatives may be therapeuticallyactive themselves or they may be prodrugs, which become active uponfurther modification. Thus, in one embodiment, a therapeutic agentderivative retains some or all of the therapeutic activity as comparedto the unmodified agent, while in another embodiment, a therapeuticagent derivative lacks therapeutic activity.

In various embodiments, therapeutic agents include any therapeuticallyeffective agent or drug, such as anti-inflammatory compounds,anti-depressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs, e.g.,anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, whichmay also be referred to as an anti-tumor drug, an anti-cancer drug, atumor drug, an antineoplastic agent, or the like. Examples of oncologydrugs that may be used according to the invention include, but are notlimited to, adriamycin, alkeran, allopurinol, altretamine, amifostine,anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU,bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda),carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin,cladribine, cyclosporin A, cytarabine, cytosine arabinoside,daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane,dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine,etoposide phosphate, etoposide and VP-16, exemestane, FK506,fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar),gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea,idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan(Camptostar, CPT-1H), letrozole, leucovorin, leustatin, leuprolide,levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna,methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone,nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin,porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,vincristine, VP16, and vinorelbine. Other examples of oncology drugsthat may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors and camptothecins.

Nucleic Acid-Lipid Particles

In certain embodiments, lipid particles of the invention are associatedwith a nucleic acid, resulting in a nucleic acid-lipid particle. Inparticular embodiments, the nucleic acid is fully encapsulated in thelipid particle. As used herein, the term “nucleic acid” is meant toinclude any oligonucleotide or polynucleotide. Fragments containing upto 50 nucleotides are generally termed oligonucleotides, and longerfragments are called polynucleotides. In particular embodiments,oligonucleotides of the invention are 20-50 nucleotides in length.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also includes polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

Oligonucleotides are classified as deoxyribooligonucleotides orribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbonsugar called deoxyribose joined covalently to phosphate at the 5′ and 3′carbons of this sugar to form an alternating, unbranched polymer. Aribooligonucleotide consists of a similar repeating structure where the5-carbon sugar is ribose.

The nucleic acid that is present in a lipid-nucleic acid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA include structural genes, genes including controland termination regions, and self-replicating systems such as viral orplasmid DNA. Examples of double-stranded RNA include siRNA and other RNAinterference reagents. Single-stranded nucleic acids include, e.g.,antisense oligonucleotides, ribozymes, microRNA, and triplex-formingoligonucleotides.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 to100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to 100 nucleotides in length.In various related embodiments, oligonucleotides, both single-stranded,double-stranded, and triple-stranded, may range in length from about 10to about 50 nucleotides, from about 20 to about 50 nucleotides, fromabout 15 to about 30 nucleotides, from about 20 to about 30 nucleotidesin length.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide. “Specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between the DNA or RNAtarget and the oligonucleotide. It is understood that an oligonucleotideneed not be 100% complementary to its target nucleic acid sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when binding of the oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of utility or expression therefrom, and there is a sufficientdegree of complementarity to avoid non-specific binding of theoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or, in the case of invitro assays, under conditions in which the assays are conducted. Thus,in other embodiments, this oligonucleotide includes 1, 2, or 3 basesubstitutions as compared to the region of a gene or mRNA sequence thatit is targeting or to which it specifically hybridizes.

RNA Interference Nucleic Acids

In particular embodiments, nucleic acid-lipid particles of the inventionare associated with RNA interference (RNAi) molecules. RNA interferencemethods using RNAi molecules may be used to disrupt the expression of agene or polynucleotide of interest. In the last 5 years smallinterfering RNA (siRNA) has essentially replaced antisense ODN andribozymes as the next generation of targeted oligonucleotide drugs underdevelopment. SiRNAs are RNA duplexes normally 21-30 nucleotides longthat can associate with a cytoplasmic multi-protein complex known asRNAi-induced silencing complex (RISC). RISC loaded with siRNA mediatesthe degradation of homologous mRNA transcripts, therefore siRNA can bedesigned to knock down protein expression with high specificity. Unlikeother antisense technologies, siRNA function through a natural mechanismevolved to control gene expression through non-coding RNA. This isgenerally considered to be the reason why their activity is more potentin vitro and in vivo than either antisense ODN or ribozymes. A varietyof RNAi reagents, including siRNAs targeting clinically relevanttargets, are currently under pharmaceutical development, as described,e.g., in de Fougerolles, A. et al., Nature Reviews 6:443-453 (2007).

While the first described RNAi molecules were RNA:RNA hybrids comprisingboth an RNA sense and an RNA antisense strand, it has now beendemonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNAantisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi(Lamberton, J. S. and Christian, A. T., (2003) Molecular Biotechnology24:111-119). Thus, the invention includes the use of RNAi moleculescomprising any of these different types of double-stranded molecules. Inaddition, it is understood that RNAi molecules may be used andintroduced to cells in a variety of forms. Accordingly, as used herein,RNAi molecules encompasses any and all molecules capable of inducing anRNAi response in cells, including, but not limited to, double-strandedpolynucleotides comprising two separate strands, i.e. a sense strand andan antisense strand, e.g., small interfering RNA (siRNA);polynucleotides comprising a hairpin loop of complementary sequences,which forms a double-stranded region, e.g., shRNAi molecules, andexpression vectors that express one or more polynucleotides capable offorming a double-stranded polynucleotide alone or in combination withanother polynucleotide.

A “single strand siRNA compound” as used herein, is an siRNA compoundwhich is made up of a single molecule. It may include a duplexed region,formed by intra-strand pairing, e.g., it may be, or include, a hairpinor pan-handle structure. Single strand siRNA compounds may be antisensewith regard to the target molecule

A single strand siRNA compound may be sufficiently long that it canenter the RISC and participate in RISC mediated cleavage of a targetmRNA. A single strand siRNA compound is at least 14, and in otherembodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides inlength. In certain embodiments, it is less than 200, 100, or 60nucleotides in length.

Hairpin siRNA compounds will have a duplex region equal to or at least17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion will may be equal to or less than 200, 100, or 50, in length. Incertain embodiments, ranges for the duplex region are 15-30, 17 to 23,19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin may havea single strand overhang or terminal unpaired region. In certainembodiments, the overhangs are 2-3 nucleotides in length. In someembodiments, the overhang is at the sense side of the hairpin and insome embodiments on the antisense side of the hairpin.

A “double stranded siRNA compound” as used herein, is an siRNA compoundwhich includes more than one, and in some cases two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded siRNA compound may be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It may be equal to or less than 200, 100, or 50, nucleotides inlength. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. As used herein, term “antisense strand” means the strand of ansiRNA compound that is sufficiently complementary to a target molecule,e.g. a target RNA.

The sense strand of a double stranded siRNA compound may be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length.It may be equal to or less than 200, 100, or 50, nucleotides in length.Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double strand portion of a double stranded siRNA compound may beequal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29,40, or 60 nucleotide pairs in length. It may be equal to or less than200, 100, or 50, nucleotides pairs in length. Ranges may be 15-30, 17 to23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the siRNA compound is sufficiently large that itcan be cleaved by an endogenous molecule, e.g., by Dicer, to producesmaller siRNA compounds, e.g., siRNAs agents

The sense and antisense strands may be chosen such that thedouble-stranded siRNA compound includes a single strand or unpairedregion at one or both ends of the molecule. Thus, a double-strandedsiRNA compound may contain sense and antisense strands, paired tocontain an overhang, e.g., one or two 5′ or 3′ overhangs, or a 3′overhang of 1-3 nucleotides. The overhangs can be the result of onestrand being longer than the other, or the result of two strands of thesame length being staggered. Some embodiments will have at least one 3′overhang. In one embodiment, both ends of an siRNA molecule will have a3′ overhang. In some embodiments, the overhang is 2 nucleotides.

In certain embodiments, the length for the duplexed region is between 15and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe ssiRNA compound range discussed above. ssiRNA compounds can resemblein length and structure the natural Dicer processed products from longdsiRNAs. Embodiments in which the two strands of the ssiRNA compound arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and a 3′ overhang are also within the invention.

The siRNA compounds described herein, including double-stranded siRNAcompounds and single-stranded siRNA compounds can mediate silencing of atarget RNA, e.g., mRNA, e.g., a transcript of a gene that encodes aprotein. For convenience, such mRNA is also referred to herein as mRNAto be silenced. Such a gene is also referred to as a target gene. Ingeneral, the RNA to be silenced is an endogenous gene or a pathogengene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs,can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an ssiRNA compound of 21 to23 nucleotides.

In one embodiment, an siRNA compound is “sufficiently complementary” toa target RNA, e.g., a target mRNA, such that the siRNA compound silencesproduction of protein encoded by the target mRNA. In another embodiment,the siRNA compound is “exactly complementary” to a target RNA, e.g., thetarget RNA and the siRNA compound anneal, for example to form a hybridmade exclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can includean internal region (e.g., of at least 10 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in certain embodiments, thesiRNA compound specifically discriminates a single-nucleotidedifference. In this case, the siRNA compound only mediates RNAi if exactcomplementary is found in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

RNA interference (RNAi) may be used to specifically inhibit expressionof target polynucleotides. Double-stranded RNA-mediated suppression ofgene and nucleic acid expression may be accomplished according to theinvention by introducing dsRNA, siRNA or shRNA into cells or organisms.SiRNA may be double-stranded RNA, or a hybrid molecule comprising bothRNA and DNA, e.g., one RNA strand and one DNA strand. It has beendemonstrated that the direct introduction of siRNAs to a cell cantrigger RNAi in mammalian cells (Elshabir, S. M., et al. Nature411:494-498 (2001)). Furthermore, suppression in mammalian cellsoccurred at the RNA level and was specific for the targeted genes, witha strong correlation between RNA and protein suppression (Caplen, N. etal., Proc. Natl. Acad. Sci. USA 98:9746-9747 (2001)). In addition, itwas shown that a wide variety of cell lines, including HeLa S3, COS7,293, NIH/3T3, A549, HT-29, CHO-KI and MCF-7 cells, are susceptible tosome level of siRNA silencing (Brown, D. et al. TechNotes 9(1):1-7,available on the worldwide web atwww.dot.ambion.dot.com/techlib/tn/91/912.html (Sep. 1, 2002)).

RNAi molecules targeting specific polynucleotides can be readilyprepared according to procedures known in the art. Structuralcharacteristics of effective siRNA molecules have been identified.Elshabir, S. M. et al. (2001) Nature 411:494-498 and Elshabir, S. M. etal. (2001), EMBO 20:6877-6888. Accordingly, one of skill in the artwould understand that a wide variety of different siRNA molecules may beused to target a specific gene or transcript. In certain embodiments,siRNA molecules according to the invention are double-stranded and 16-30or 18-25 nucleotides in length, including each integer in between. Inone embodiment, an siRNA is 21 nucleotides in length. In certainembodiments, siRNAs have 0-7 nucleotide 3′ overhangs or 0-4 nucleotide5′ overhangs. In one embodiment, an siRNA molecule has a two nucleotide3′ overhang. In one embodiment, an siRNA is 21 nucleotides in lengthwith two nucleotide 3′ overhangs (i.e. they contain a 19 nucleotidecomplementary region between the sense and antisense strands). Incertain embodiments, the overhangs are UU or dTdT 3′ overhangs.

Generally, siRNA molecules are completely complementary to one strand ofa target DNA molecule, since even single base pair mismatches have beenshown to reduce silencing. In other embodiments, siRNAs may have amodified backbone composition, such as, for example, 2′-deoxy- or2′-O-methyl modifications. However, in preferred embodiments, the entirestrand of the siRNA is not made with either 2′ deoxy or 2′-O-modifiedbases.

In another embodiment, the invention provides a cell including a vectorfor inhibiting the expression of a gene in a cell. The vector includes aregulatory sequence operably linked to a nucleotide sequence thatencodes at least one strand of one of the dsRNA of the invention.

In one embodiment, siRNA target sites are selected by scanning thetarget mRNA transcript sequence for the occurrence of AA dinucleotidesequences. Each AA dinucleotide sequence in combination with the 3′adjacent approximately 19 nucleotides are potential siRNA target sites.In one embodiment, siRNA target sites are preferentially not locatedwithin the 5′ and 3′ untranslated regions (UTRs) or regions near thestart codon (within approximately 75 bases), since proteins that bindregulatory regions may interfere with the binding of the siRNPendonuclease complex (Elshabir, S. et al. Nature 411:494-498 (2001);Elshabir, S. et al. EMBO J. 20:6877-6888 (2001)). In addition, potentialtarget sites may be compared to an appropriate genome database, such asBLASTN 2.0.5, available on the NCBI server at www.ncbi.nlm, andpotential target sequences with significant homology to other codingsequences eliminated.

In particular embodiments, short hairpin RNAs constitute the nucleicacid component of nucleic acid-lipid particles of the invention. ShortHairpin RNA (shRNA) is a form of hairpin RNA capable ofsequence-specifically reducing expression of a target gene. Shorthairpin RNAs may offer an advantage over siRNAs in suppressing geneexpression, as they are generally more stable and less susceptible todegradation in the cellular environment. It has been established thatsuch short hairpin RNA-mediated gene silencing works in a variety ofnormal and cancer cell lines, and in mammalian cells, including mouseand human cells. Paddison, P. et al., Genes Dev. 16(8):948-58 (2002).Furthermore, transgenic cell lines bearing chromosomal genes that codefor engineered shRNAs have been generated. These cells are able toconstitutively synthesize shRNAs, thereby facilitating long-lasting orconstitutive gene silencing that may be passed on to progeny cells.Paddison, P. et al., Proc. Natl. Acad. Sci. USA 99(3):1443-1448 (2002).

ShRNAs contain a stem loop structure. In certain embodiments, they maycontain variable stem lengths, typically from 19 to 29 nucleotides inlength, or any number in between. In certain embodiments, hairpinscontain 19 to 21 nucleotide stems, while in other embodiments, hairpinscontain 27 to 29 nucleotide stems. In certain embodiments, loop size isbetween 4 to 23 nucleotides in length, although the loop size may belarger than 23 nucleotides without significantly affecting silencingactivity. ShRNA molecules may contain mismatches, for example G-Umismatches between the two strands of the shRNA stem without decreasingpotency. In fact, in certain embodiments, shRNAs are designed to includeone or several G-U pairings in the hairpin stem to stabilize hairpinsduring propagation in bacteria, for example. However, complementaritybetween the portion of the stem that binds to the target mRNA (antisensestrand) and the mRNA is typically required, and even a single base pairmismatch is this region may abolish silencing. 5′ and 3′ overhangs arenot required, since they do not appear to be critical for shRNAfunction, although they may be present (Paddison et al. (2002) Genes &Dev. 16(8):948-58).

MicroRNAs

Micro RNAs (miRNAs) are a highly conserved class of small RNA moleculesthat are transcribed from DNA in the genomes of plants and animals, butare not translated into protein. Processed miRNAs are singlestranded-17-25 nucleotide (nt) RNA molecules that become incorporatedinto the RNA-induced silencing complex (RISC) and have been identifiedas key regulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in: “miRBase:microRNA sequences, targets and gene nomenclature” Griffiths-Jones S,Grocock R J, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34,Database Issue, D140-D144; “The microRNA Registry” Griffiths-Jones S,NAR, 2004, 32, Database Issue, D109-D111; and also on the worldwide webat microrna.dot.sanger.dot.ac.dot.uk/sequences/.

Antisense Oligonucleotides

In one embodiment, a nucleic acid is an antisense oligonucleotidedirected to a target polynucleotide. The term “antisenseoligonucleotide” or simply “antisense” is meant to includeoligonucleotides that are complementary to a targeted polynucleotidesequence. Antisense oligonucleotides are single strands of DNA or RNAthat are complementary to a chosen sequence. In the case of antisenseRNA, they prevent translation of complementary RNA strands by binding toit. Antisense DNA can be used to target a specific, complementary(coding or non-coding) RNA. If binding takes places this DNA/RNA hybridcan be degraded by the enzyme RNase H. In particular embodiment,antisense oligonucleotides contain from about 10 to about 50nucleotides, more preferably about 15 to about 30 nucleotides. The termalso encompasses antisense oligonucleotides that may not be exactlycomplementary to the desired target gene. Thus, the invention can beutilized in instances where non-target specific-activities are foundwith antisense, or where an antisense sequence containing one or moremismatches with the target sequence is the most preferred for aparticular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examplesof antisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989; 1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998Jun. 15; 57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Furthermore,antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. Antagomirs may be usedto efficiently silence endogenous miRNAs by forming duplexes comprisingthe antagomir and endogenous miRNA, thereby preventing miRNA-inducedgene silencing. An example of antagomir-mediated miRNA silencing is thesilencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438:685-689, which is expressly incorporated by reference herein in itsentirety. Antagomir RNAs may be synthesized using standard solid phaseoligonucleotide synthesis protocols.'See U.S. patent application Ser.Nos. 11/502,158 and 11/657,341 (the disclosure of each of which areincorporated herein by reference).

An antagomir can include ligand-conjugated monomer subunits and monomersfor oligonucleotide synthesis. Exemplary monomers are described in U.S.application Ser. No. 10/916,185, filed on Aug. 10, 2004. An antagomircan have a ZXY structure, such as is described in PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004. An antagomir can be complexedwith an amphipathic moiety. Exemplary amphipathic moieties for use witholigonucleotide agents are described in PCT Application No.PCT/US2004/07070, filed on Mar. 8, 2004.

Aptamers

Aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).DNA or RNA aptamers have been successfully produced which bind manydifferent entities from large proteins to small organic molecules. SeeEaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin.Struct. Biol. 9:324-9 (1999), and Hermann and Patel, Science 287:820-5(2000). Aptamers may be RNA or DNA based, and may include a riboswitch.A riboswitch is a part of an mRNA molecule that can directly bind asmall target molecule, and whose binding of the target affects thegene's activity. Thus, an mRNA that contains a riboswitch is directlyinvolved in regulating its own activity, depending on the presence orabsence of its target molecule. Generally, aptamers are engineeredthrough repeated rounds of in vitro selection or equivalently, SELEX(systematic evolution of ligands by exponential enrichment) to bind tovarious molecular targets such as small molecules, proteins, nucleicacids, and even cells, tissues and organisms. The aptamer may beprepared by any known method, including synthetic, recombinant, andpurification methods, and may be used alone or in combination with otheraptamers specific for the same target. Further, as described more fullyherein, the term “aptamer” specifically includes “secondary aptamers”containing a consensus sequence derived from comparing two or more knownaptamers to a given target.

Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24; 49(2):211-20).For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., Cell. 1981 December; 27(3 Pt 2):487-96; Michel and Westhof, JMol. Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature.1992 May 14; 357(6374):173-6). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif, forexample. Specific examples of hammerhead motifs are described by Rossiet al. Nucleic Acids Res. 1992 Sep. 11; 20(17):4559-65. Examples ofhairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No.EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan. 25;18(2):299-304 and U.S. Pat. No. 5,631,359. An example of the hepatitis δvirus motif is described by Perrotta and Been, Biochemistry. 1992 Dec.1; 31(47):11843-52; an example of the RNaseP motif is described byGuerrier-Takada et al., Cell. 1983 December; 35(3 Pt 2):849-57;Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins, Proc NatlAcad Sci USA. 1991 Oct. 1; 88(19):8826-30; Collins and Olive,Biochemistry. 1993 Mar. 23; 32(11):2795-9); and an example of the GroupI intron is described in U.S. Pat. No. 4,987,071. Importantcharacteristics of enzymatic nucleic acid molecules used according tothe invention are that they have a specific substrate binding site whichis complementary to one or more of the target gene DNA or RNA regions,and that they have nucleotide sequences within or surrounding thatsubstrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in Int.Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO94/02595, each specifically incorporated herein by reference, andsynthesized to be tested in vitro and in vivo, as described therein.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem H bases toshorten RNA synthesis times and reduce chemical requirements.

Additional specific nucleic acid sequences of oligonucleotides (ODNs)suitable for use in the compositions and methods of the invention aredescribed in U.S. Patent Appln. 60/379,343, U.S. patent application Ser.No. 09/649,527, Int. Publ. WO 02/069369, Int. Publ. No. WO 01/15726,U.S. Pat. No. 6,406,705, and Raney et al., Journal of Pharmacology andExperimental Therapeutics, 298:1185-1192 (2001). In certain embodiments,ODNs used in the compositions and methods of the invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

Nucleic Acid Modifications

In the 1990's DNA-based antisense oligodeoxynucleotides (ODN) andribozymes (RNA) represented an exciting new paradigm for drug design anddevelopment, but their application in vivo was prevented by endo- andexo-nuclease activity as well as a lack of successful intracellulardelivery. The degradation issue was effectively overcome followingextensive research into chemical modifications that prevented theoligonucleotide (oligo) drugs from being recognized by nuclease enzymesbut did not inhibit their mechanism of action. This research was sosuccessful that antisense ODN drugs in development today remain intactin vivo for days compared to minutes for unmodified molecules (Kurreck,J. 2003. Antisense technologies. Improvement through novel chemicalmodifications. Eur J Biochem 270:1628-44). However, intracellulardelivery and mechanism of action issues have so far limited antisenseODN and ribozymes from becoming clinical products.

RNA duplexes are inherently more stable to nucleases than singlestranded DNA or RNA, and unlike antisense ODN, unmodified siRNA showgood activity once they access the cytoplasm. Even so, the chemicalmodifications developed to stabilize antisense ODN and ribozymes havealso been systematically applied to siRNA to determine how much chemicalmodification can be tolerated and if pharmacokinetic and pharmacodynamicactivity can be enhanced. RNA interference by siRNA duplexes requires anantisense and sense strand, which have different functions. Both arenecessary to enable the siRNA to enter RISC, but once loaded the twostrands separate and the sense strand is degraded whereas the antisensestrand remains to guide RISC to the target mRNA. Entry into RISC is aprocess that is structurally less stringent than the recognition andcleavage of the target mRNA. Consequently, many different chemicalmodifications of the sense strand are possible, but only limited changesare tolerated by the antisense strand (Zhang et al., 2006).

As is known in the art, a nucleoside is a base-sugar combination.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turnthe respective ends of this linear polymeric structure can be furtherjoined to form a circular structure. Within the oligonucleotidestructure, the phosphate groups are commonly referred to as forming theinternucleoside backbone of the oligonucleotide. The normal linkage orbackbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

The nucleic acid that is used in a lipid-nucleic acid particle accordingto this invention includes any form of nucleic acid that is known. Thus,the nucleic acid may be a modified nucleic acid of the type usedpreviously to enhance nuclease resistance and serum stability.Surprisingly, however, acceptable therapeutic products can also beprepared using the method of the invention to formulate lipid-nucleicacid particles from nucleic acids that have no modification to thephosphodiester linkages of natural nucleic acid polymers, and the use ofunmodified phosphodiester nucleic acids (i.e., nucleic acids in whichall of the linkages are phosphodiester linkages) is a preferredembodiment of the invention.

Backbone Modifications

Antisense, siRNA and other oligonucleotides useful in this inventioninclude, but are not limited to, oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified oligonucleotides that do not have a phosphorus atomin their internucleoside backbone can also be considered to beoligonucleosides. Modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, phosphoroselenoate, methylphosphonate, orO-alkyl phosphotriester linkages, and boranophosphates having normal3′-5′ linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Particular non-limiting examples ofparticular modifications that may be present in a nucleic acid accordingto the invention are shown in Table 2.

Various salts, mixed salts and free acid forms are also included.Representative United States patents that teach the preparation of theabove linkages include, but are not limited to, U.S. Pat. Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050.

In certain embodiments, modified oligonucleotide backbones that do notinclude a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include, e.g., those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.Representative United States patents that describe the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439.

The phosphorothioate backbone modification (Table 3, #1), where anon-bridging oxygen in the phosphodiester bond is replaced by sulfur, isone of the earliest and most common means deployed to stabilize nucleicacid drugs against nuclease degradation. In general, it appears that PSmodifications can be made extensively to both siRNA strands without muchimpact on activity (Kurreck, J., Eur. J. Biochem. 270:1628-44, 2003).However, PS oligos are known to avidly associate non-specifically withproteins resulting in toxicity, especially upon i.v. administration.Therefore, the PS modification is usually restricted to one or two basesat the 3′ and 5′ ends. The boranophosphate linker (Table 3, #2) is arecent modification that is apparently more stable than PS, enhancessiRNA activity and has low toxicity (Hall et al., Nucleic Acids Res.32:5991-6000, 2004).

TABLE 3 Chemical Modifications Applied to siRNA and Other Nucleic AcidsAbbrev- Modification # iation Name Site Structure 1 PS PhosphorothioateBackbone

2 PB Boranophosphate Backbone

3 N3-MU N3-methyl- uridine Base

4 5′-BU 5′-bromo-uracil Base

5 5′-IU 5′-iodo-uracil Base

6 2,6-DP 2,6- diaminopurine Base

7 2′-F 2′-Fluoro Sugar

8 2′-OME 2″-O-methyl Sugar

9 2′-O-MOE 2′-O-(2- methoxylethyl) Sugar

10 2′-DNP 2′-O-(2,4- dinitrophenyl) Sugar

11 LNA Locked Nucleic Acid (methylene bridge connecting the 2′- oxygenwith the 4′-carbon of the ribose ring) Sugar

12 2′-Amino 2′-Amino Sugar

13 2′-Deoxy 2′-Deoxy Sugar

14 4′-thio 4′-thio- ribonucleotide Sugar

Other useful nucleic acids derivatives include those nucleic acidsmolecules in which the bridging oxygen atoms (those forming thephosphoester linkages) have been replaced with —S—, —NH—, —CH2- and thelike. In certain embodiments, the alterations to the antisense, siRNA,or other nucleic acids used will not completely affect the negativecharges associated with the nucleic acids. Thus, the inventioncontemplates the use of antisense, siRNA, and other nucleic acids inwhich a portion of the linkages are replaced with, for example, theneutral methyl phosphonate or phosphoramidate linkages. When neutrallinkages are used, in certain embodiments, less than 80% of the nucleicacid linkages are so substituted, or less than 50% of the linkages areso substituted.

Base Modifications

Base modifications are less common than those to the backbone and sugar.The modifications shown in 0.3-6 all appear to stabilize siRNA againstnucleases and have little effect on activity (Zhang, H. Y., Du, Q.,Wahlestedt, C., Liang, Z. 2006. RNA Interference with chemicallymodified siRNA. Curr Top Med Chem 6:893-900).

Accordingly, oligonucleotides may also include nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C orm5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Certain nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention, including5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications 1993, CRC Press, Boca Raton, pages 276-278). These may becombined, in particular embodiments, with 2′-O-methoxyethyl sugarmodifications. United States patents that teach the preparation ofcertain of these modified nucleobases as well as other modifiednucleobases include, but are not limited to, the above noted U.S. Pat.No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941.

Sugar Modifications

Most modifications on the sugar group occur at the 2′-OH of the RNAsugar ring, which provides a convenient chemically reactive site(Manoharan, M. 2004. RNA interference and chemically modified smallinterfering RNAs. Curr Opin Chem Biol 8:570-9; Zhang, H. Y., Du, Q.,Wahlestedt, C., Liang, Z. 2006. RNA Interference with chemicallymodified siRNA. Curr Top Med Chem 6:893-900). The 2′-F and 2% OME (0.7and 8) are common and both increase stability, the 2′-OME modificationdoes not reduce activity as long as it is restricted to less than 4nucleotides per strand (Holen, T., Amarzguioui, M., Babaie, E., Prydz,H. 2003. Similar behaviour of single-strand and double-strand siRNAssuggests they act through a common RNAi pathway. Nucleic Acids Res31:2401-7). The 2′-O-MOE (0.9) is most effective in siRNA when modifiedbases are restricted to the middle region of the molecule (Prakash, T.P., Allerson, C. R., Dande, P., Vickers, T. A., Sioufi, N., Janes, R.,Baker, B. F., Swayze, E. E., Griffey, R. H., Bhat, B. 2005. Positionaleffect of chemical modifications on short interference RNA activity inmammalian cells. J Med Chem 48:4247-53). Other modifications found tostabilize siRNA without loss of activity are shown in 0.10-14.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. For example, the invention includes oligonucleotides thatcomprise one of the following at the 2′ position: OH; F; O-, S-, orN-alkyl, O-alkyl-O-alkyl, O-, S-, or N-alkenyl, or O-, S- or N-alkynyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C2 to C10 alkenyl and alkynyl.Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)₂ON(CH₃)₂, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. One modification includes2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504), i.e., analkoxyalkoxy group. Other modifications include2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (2′-DMAEOE).

Additional modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarsstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and5,700,920.

In other oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups, although the base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al. (Science, 1991, 254,1497-1500).

Particular embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—(referred to as a methylene(methylimino) or MMI backbone)—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—O—N(CH₃)—CH₂—CH₂—(wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—) of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, an oligonucleotide can include nucleotidescontaining e.g., arabinose, as the sugar. The monomer can have an alphalinkage at the 1′ position on the sugar, e.g., alpha-nucleosides.Oligonucleotides can also include “abasic” sugars, which lack anucleobase at C-1′. These abasic sugars can also be further containingmodifications at one or more of the constituent sugar atoms.Oligonucleotides can also contain one or more sugars that are in the Lform, e.g. L-nucleosides.

Chimeric Oligonucleotides

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. Certain preferredoligonucleotides of this invention are chimeric oligonucleotides.“Chimeric oligonucleotides” or “chimeras,” in the context of thisinvention, are oligonucleotides that contain two or more chemicallydistinct regions, each made up of at least one nucleotide. Theseoligonucleotides typically contain at least one region of modifiednucleotides that confers one or more beneficial properties (such as,e.g., increased nuclease resistance, increased uptake into cells,increased binding affinity for the RNA target) and a region that is asubstrate for RNase H cleavage.

In one embodiment, a chimeric oligonucleotide comprises at least oneregion modified to increase target binding affinity. Affinity of anoligonucleotide for its target is routinely determined by measuring theTm of an oligonucleotide/target pair, which is the temperature at whichthe oligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target. In one embodiment, the region of theoligonucleotide which is modified to increase target mRNA bindingaffinity comprises at least one nucleotide modified at the 2′ positionof the sugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance oligonucleotide inhibition oftarget gene expression.

In another embodiment, a chimeric oligonucleotide comprises a regionthat acts as a substrate for RNAse H. Of course, it is understood thatoligonucleotides may include any combination of the variousmodifications described herein.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such conjugates and methods of preparingthe same are known in the art.

Those skilled in the art will realize that for in vivo utility, such astherapeutic efficacy, a reasonable rule of thumb is that if a thioatedversion of the sequence works in the free form, that encapsulatedparticles of the same sequence, of any chemistry, will also beefficacious. Encapsulated particles may also have a broader range of invivo utilities, showing efficacy in conditions and models not known tobe otherwise responsive to antisense therapy. Those skilled in the artknow that applying this invention they may find old models which nowrespond to antisense therapy. Further, they may revisit discardedantisense sequences or chemistries and find efficacy by employing theinvention.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal or other patient.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076),or CpG motifs, as well as other known ISS features (such as multi-Gdomains, see WO 96/11266).

The immune response may be an innate or an adaptive immune response. Theimmune system is divided into a more innate immune system, and acquiredadaptive immune system of vertebrates, the latter of which is furtherdivided into humoral cellular components. In particular embodiments, theimmune response may be mucosal.

In particular embodiments, an immunostimulatory nucleic acid is onlyimmunostimulatory when administered in combination with a lipidparticle, and is not immunostimulatory when administered in its “freeform.” According to the present invention, such an oligonucleotide isconsidered to be immunostimulatory.

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target polynucleotide in order to provoke animmune response. Thus, certain immunostimulatory nucleic acids maycomprise a sequence corresponding to a region of a naturally occurringgene or mRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein said CpG dinucleotide is methylated. In a specific embodiment, thenucleic acid comprises the sequence 5′ TAACGTTGAGGGGCAT 3′. In analternative embodiment, the nucleic acid comprises at least two CpGdinucleotides, wherein at least one cytosine in the CpG dinucleotides ismethylated. In a further embodiment, each cytosine in the CpGdinucleotides present in the sequence is methylated. In anotherembodiment, the nucleic acid comprises a plurality of CpG dinucleotides,wherein at least one of said CpG dinucleotides comprises a methylatedcytosine.

In one specific embodiment, the nucleic acid comprises the sequence 5′TTCCATGACGTTCCTGACGT 3′. In another specific embodiment, the nucleicacid sequence comprises the sequence 5′ TCCATGACGTTCCTGACGT 3′, whereinthe two cytosines indicated in bold are methylated. In particularembodiments, the ODN is selected from a group of ODNs consisting of ODN#1, ODN #2, ODN #3, ODN #4, ODN #5, ODN #6, ODN #7, ODN #8, and ODN #9,as shown below.

TABLE 4  Exemplary Immunostimulatory Oligonucleotides (ODNs) ODN NAMEEQ ID ODN SEQUENCE (5′-3′) ODN 1 human c-myc 5′-TAACGTTGAGGGGCAT-3* ODN 1m 5′-TAAZGTTGAGGGGCAT-3 ODN 2 5′-TCCATGACGTTCCTGACGTT-3 * ODN 2m5′-TCCATGAZGTTCCTGAZGTT-3 ODN 3 5′-TAAGCATACGGGGTGT-3 ODN 5 5′-AACGTT-3ODN 6 5′-GATGCTGTGTCGGGGTCTCCGGGC-3′ ODN 75′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ ODN 7m 5′-TZGTZGTTTTGTZGTTTTGTZGTT-3′ODN 8 15′-TCCAGGACTTCTCTCAGGTT-3′ ODN 9 5′-TCTCCCAGCGTGCGCCAT-3′ODN 10 murine Intracellular 5′-TGCATCCCCCAGGCCACCAT-3Adhesion Molecule-1 ODN 11 human Intracellular5′-GCCCAAGCTGGCATCCGTCA-3′ Adhesion Molecule-1ODN 12 human Intracellular 5′-GCCCAAGCTGGCATCCGTCA-3′Adhesion Molecule-1 ODN 13 human erb-B-2 5′-GGT GCTCACTGC GGC-3′ODN 14 human c-myc 5′-AACC GTT GAG GGG CAT-3′ ODN 15 human c-myc5′-TAT GCT GTG CCG GGG TCT TCG GGC-3′ ODN 16 5′-GTGCCG GGGTCTTCGGGC-3′ODN 17 human Insulin 5′-GGACCCTCCTCCGGAGCC-3′ Growth Factor 1-ReceptorODN 18 human Insulin 5′-TCC TCC GGA GCC AGA CTT-3′Growth Factor 1-Receptor ODN 19 human Epidermal5′-AAC GTT GAG GGG CAT-3′ Growth Factor-Receptor ODN 20 Epidermal Growth5′-CCGTGGTCA TGCTCC-3′ Factor-Receptor ODN 21 human Vascular5′-CAG CCTGGCTCACCG CCTTGG-3′ Endothelial Growth Factor ODN 22 murine5′-CAG CCA TGG TTC CCC CCA AC-3′ Phosphokinase C-alpha ODN 235′-GTT CTC GCT GGT GAG TTT CA-3′ ODN 24 human Bcl-25′-TCT CCCAGCGTGCGCCAT-3′ ODN 25 human C-Raf-s 5′-GTG CTC CAT TGA TGC-3′ODN #26 human Vascular 5′-GAGUUCUGAUGAGGCCGAAAGG- Endothelial GrowthCCGAAAGUCUG-3′ Factor Receptor-1 ODN #27 5′-RRCGYY-3′ ODN #285′-AACGTTGAGGGGCAT-3′ ODN #29 5′-CAACGTTATGGGGAGA-3′ ODN #30 human c-myc5′-TAACGTTGAGGGGCAT-3′ “Z” represents a methylated cytosine residue. ODN14 is a 15-mer oligonucleotide and ODN 1 is the same oligonucleotidehaving a thymidine added onto the 5′ end making ODN 1 into a 16-mer. Nodifference in biological activity between ODN 14 and ODN 1 has beendetected and both exhibit similar immunostimulatory activity (Mui etal., 2001)

Additional specific nucleic acid sequences of oligonucleotides (ODNs)suitable for use in the compositions and methods of the invention aredescribed in Raney et al., Journal of Pharmacology and ExperimentalTherapeutics, 298:1185-1192 (2001). In certain embodiments, ODNs used inthe compositions and methods of the present invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

Decoy Oligonucleotides

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides may be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety

Supermir

A supermir refers to a single stranded, double stranded or partiallydouble stranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or both or modifications thereof, which hasa nucleotide sequence that is substantially identical to an miRNA andthat is antisense with respect to its target. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages and which contain at leastone non-naturally-occurring portion which functions similarly. Suchmodified or substituted oligonucleotides are preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. In a preferred embodiment, thesupermir does not include a sense strand, and in another preferredembodiment, the supermir does not self-hybridize to a significantextent. An supermir featured in the invention can have secondarystructure, but it is substantially single-stranded under physiologicalconditions. An supermir that is substantially single-stranded issingle-stranded to the extent that less than about 50% (e.g., less thanabout 40%, 30%, 20%, 10%, or 5%) of the supermir is duplexed withitself. The supermir can include a hairpin segment, e.g., sequence,preferably at the 3′ end can self hybridize and form a duplex region,e.g., a duplex region of at least 1, 2, 3, or 4 and preferably less than8, 7, 6, or n nucleotides, e.g., 5 nucleotides. The duplexed region canbe connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or 6dTs, e.g., modified dTs. In another embodiment the supermir is duplexedwith a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10 nucleotides inlength, e.g., at one or both of the 3′ and 5′ end or at one end and inthe non-terminal or middle of the supermir.

miRNA Mimics

miRNA mimics represent a class of molecules that can be used to imitatethe gene silencing ability of one or more miRNAs. Thus, the term“microRNA mimic” refers to synthetic non-coding RNAs (i.e. the miRNA isnot obtained by purification from a source of the endogenous miRNA) thatare capable of entering the RNAi pathway and regulating gene expression.miRNA mimics can be designed as mature molecules (e.g. single stranded)or mimic precursors (e.g., pri- or pre-miRNAs). miRNA mimics can becomprised of nucleic acid (modified or modified nucleic acids) includingoligonucleotides comprising, without limitation, RNA, modified RNA, DNA,modified DNA, locked nucleic acids, or 2′-O,4′-C-ethylene-bridgednucleic acids (ENA), or any combination of the above (including DNA-RNAhybrids). In addition, miRNA mimics can comprise conjugates that canaffect delivery, intracellular compartmentalization, stability,specificity, functionality, strand usage, and/or potency. In one design,miRNA mimics are double stranded molecules (e.g., with a duplex regionof between about 16 and about 31 nucleotides in length) and contain oneor more sequences that have identity with the mature strand of a givenmiRNA. Modifications can comprise 2′ modifications (including 2′-Omethyl modifications and 2′ F modifications) on one or both strands ofthe molecule and internucleotide modifications (e.g. phosphorthioatemodifications) that enhance nucleic acid stability and/or specificity.In addition, miRNA mimics can include overhangs. The overhangs canconsist of 1-6 nucleotides on either the 3′ or 5′ end of either strandand can be modified to enhance stability or functionality. In oneembodiment, a miRNA mimic comprises a duplex region of between 16 and 31nucleotides and one or more of the following chemical modificationpatterns: the sense strand contains 2′-O-methyl modifications ofnucleotides 1 and 2 (counting from the 5′ end of the senseoligonucleotide), and all of the Cs and Us; the antisense strandmodifications can comprise 2′ F modification of all of the Cs and Us,phosphorylation of the 5′ end of the oligonucleotide, and stabilizedinternucleotide linkages associated with a 2 nucleotide 3′ overhang.

Antimir or miRNA Inhibitor.

The terms “antimir” “microRNA inhibitor”, “miR inhibitor”, or“inhibitor” are synonymous and refer to oligonucleotides or modifiedoligonucleotides that interfere with the ability of specific miRNAs. Ingeneral, the inhibitors are nucleic acid or modified nucleic acids innature including oligonucleotides comprising RNA, modified RNA, DNA,modified DNA, locked nucleic acids (LNAs), or any combination of theabove. Modifications include 2′ modifications (including 2′-0 alkylmodifications and 2′ F modifications) and internucleotide modifications(e.g. phosphorothioate modifications) that can affect delivery,stability, specificity, intracellular compartmentalization, or potency.In addition, miRNA inhibitors can comprise conjugates that can affectdelivery, intracellular compartmentalization, stability, and/or potency.Inhibitors can adopt a variety of configurations including singlestranded, double stranded (RNA/RNA or RNA/DNA duplexes), and hairpindesigns, in general, microRNA inhibitors comprise contain one or moresequences or portions of sequences that are complementary or partiallycomplementary with the mature strand (or strands) of the miRNA to betargeted, in addition, the miRNA inhibitor may also comprise additionalsequences located 5′ and 3′ to the sequence that is the reversecomplement of the mature miRNA. The additional sequences may be thereverse complements of the sequences that are adjacent to the maturemiRNA in the pri-miRNA from which the mature miRNA is derived, or theadditional sequences may be arbitrary sequences (having a mixture of A,G, C, or U). In some embodiments, one or both of the additionalsequences are arbitrary sequences capable of forming hairpins. Thus, insome embodiments, the sequence that is the reverse complement of themiRNA is flanked on the 5′ side and on the 3′ side by hairpinstructures. Micro-RNA inhibitors, when double stranded, may includemismatches between nucleotides on opposite strands. Furthermore,micro-RNA inhibitors may be linked to conjugate moieties in order tofacilitate uptake of the inhibitor into a cell. For example, a micro-RNAinhibitor may be linked to cholesteryl5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate) whichallows passive uptake of a micro-RNA inhibitor into a cell. Micro-RNAinhibitors, including hairpin miRNA inhibitors, are described in detailin Vermeulen et al., “Double-Stranded Regions Are Essential DesignComponents Of Potent Inhibitors of RISC Function,” RNA 13: 723-730(2007) and in WO2007/095387 and WO 2008/036825 each of which isincorporated herein by reference in its entirety. A person of ordinaryskill in the art can select a sequence from the database for a desiredmiRNA and design an inhibitor useful for the methods disclosed herein.

U1 Adaptor

U1 adaptor inhibit polyA sites and are bifunctional oligonucleotideswith a target domain complementary to a site in the target gene'sterminal exon and a ‘U1 domain’ that binds to the U1 smaller nuclear RNAcomponent of the U1 snRNP (Goraczniak, et al., 2008, NatureBiotechnology, 27(3), 257-263, which is expressly incorporated byreference herein, in its entirety). U1 snRNP is a ribonucleoproteincomplex that functions primarily to direct early steps in spliceosomeformation by binding to the pre-mRNA exon-intron boundary (Brown andSimpson, 1998, Annu Rev Plant Physiol Plant Mol Biol 49:77-95).Nucleotides 2-11 of the 5′ end of U1 snRNA base pair bind with the 5′ssof the pre mRNA. In one embodiment, oligonucleotides of the inventionare U1 adaptors. In one embodiment, the U1 adaptor can be administeredin combination with at least one other iRNA agent.

Oligonucleotide Modifications

Unmodified oligonucleotides may be less than optimal in someapplications, e.g., unmodified oligonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications ofoligonucleotides can confer improved properties, and, e.g., can renderoligonucleotides more stable to nucleases.

As oligonucleotides are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin an oligonucleotide, e.g., a modification of a base, a sugar, aphosphate moiety, or the non-bridging oxygen of a phosphate moiety. Itis not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even ata single nucleoside within an oligonucleotide.

In some cases the modification will occur at all of the subjectpositions in the oligonucleotide but in many, and in fact in most casesit will not. By way of example, a modification may only occur at a 3′ or5′ terminal position, may only occur in the internal region, may onlyoccur in a terminal regions, e.g. at a position on a terminal nucleotideor in the last 2, 3, 4, 5, or 10 nucleotides of an oligonucleotide. Amodification may occur in a double strand region, a single strandregion, or in both.

A modification may occur only in the double strand region of adouble-stranded oligonucleotide or may only occur in a single strandregion of a double-stranded oligonucleotide. E.g., a phosphorothioatemodification at a non-bridging oxygen position may only occur at one orboth termini, may only occur in a terminal regions, e.g., at a positionon a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides ofa strand, or may occur in double strand and single strand regions,particularly at termini. The 5′ end or ends can be phosphorylated.

A modification described herein may be the sole modification, or thesole type of modification included on multiple nucleotides, or amodification can be combined with one or more other modificationsdescribed herein. The modifications described herein can also becombined onto an oligonucleotide, e.g. different nucleotides of anoligonucleotide have different modifications described herein.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular nucleobases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-bridging oxygen atoms. However, thephosphate group can be modified by replacing one of the oxygens with adifferent substituent. One result of this modification to RNA phosphatebackbones can be increased resistance of the oligoribonucleotide tonucleolytic breakdown. Thus while not wishing to be bound by theory, itcan be desirable in some embodiments to introduce alterations whichresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoramidates, alkyl or aryl phosphonates andphosphotriesters. In certain embodiments, one of the non-bridgingphosphate oxygen atoms in the phosphate backbone moiety can be replacedby any of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl), C(i.e. an alkyl group, an aryl group, etc. . . . ), H, NR₂ (R ishydrogen, alkyl, aryl), or OR (R is alkyl or aryl). The phosphorous atomin an unmodified phosphate group is achiral. However, replacement of oneof the non-bridging oxygens with one of the above atoms or groups ofatoms renders the phosphorous atom chiral; in other words a phosphorousatom in a phosphate group modified in this way is a stereogenic center.The stereogenic phosphorous atom can possess either the “R”configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligoribonucleotides diastereomers. Thus,while not wishing to be bound by theory, modifications to bothnon-bridging oxygens, which eliminate the chiral center, e.g.phosphorodithioate formation, may be desirable in that they cannotproduce diastereomer mixtures. Thus, the non-bridging oxygens can beindependently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon is preferred. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen is preferred.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. While not wishing to be bound by theory, it is believed thatsince the charged phosphodiester group is the reaction center innucleolytic degradation, its replacement with neutral structural mimicsshould impart enhanced nuclease stability. Again, while not wishing tobe bound by theory, it can be desirable, in some embodiment, tointroduce alterations in which the charged phosphate group is replacedby a neutral moiety.

Examples of moieties which can replace the phosphate group includemethyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include themethylenecarbonylamino and methylenemethylimino groups.

Modified phosphate linkages where at least one of the oxygens linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non phosphodiesterbackbone linkage.”

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiment, to introduce alterations in which thebases are tethered by a neutral surrogate backbone. Examples include themorpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA)nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a linker. The terminal atom of the linker canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the linkercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs).

When a linker/phosphate-functional molecular entity-linker/phosphatearray is interposed between two strands of a dsRNA, this array cansubstitute for a hairpin RNA loop in a hairpin-type RNA agent.

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. E.g., inpreferred embodiments antisense strands of dsRNAs, are 5′ phosphorylatedor include a phosphoryl analog at the 5′ prime terminus. 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)₂(O)P—O-5); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5); 5′-adenosine cap (Appp),and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5); 5′-monothiophosphate(phosphorothioate; (HO)₂(S)P—O-5); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorescein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Nucleobases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nebularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases”, “modified bases”, “non-natural bases” and“universal bases” described herein, can be employed. Examples includewithout limitation 2-aminoadenine, 6-methyl and other alkyl derivativesof adenine and guanine, 2-propyl and other alkyl derivatives of adenineand guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Cationic Groups

Modifications to oligonucleotides can also include attachment of one ormore cationic groups to the sugar, base, and/or the phosphorus atom of aphosphate or modified phosphate backbone moiety. A cationic group can beattached to any atom capable of substitution on a natural, unusual oruniversal base. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Placement within an Oligonucleotide

Some modifications may preferably be included on an oligonucleotide at aparticular location, e.g., at an internal position of a strand, or onthe 5′ or 3′ end of an oligonucleotide. A preferred location of amodification on an oligonucleotide, may confer preferred properties onthe agent. For example, preferred locations of particular modificationsmay confer optimum gene silencing properties, or increased resistance toendonuclease or exonuclease activity.

One or more nucleotides of an oligonucleotide may have a 2′-5′ linkage.One or more nucleotides of an oligonucleotide may have invertedlinkages, e.g. 3′-3′, 5′-5′,2′-2′ or 2′-3′ linkages.

A double-stranded oligonucleotide may include at least one5′-uridine-adenine-3′ (5′-UG-3′) dinucleotide wherein the uridine is a2′-modified nucleotide, or a terminal 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide, or aterminal 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide, or a terminal5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide, or a terminal 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide, or a terminal 5′-cytidine-uridine-3′ (5′-CU-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, or aterminal 5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide. Double-stranded oligonucleotidesincluding these modifications are particularly stabilized againstendonuclease activity.

General References

The oligoribonucleotides and oligoribonucleotides used in accordancewith this invention may be synthesized with solid phase synthesis, seefor example “Oligonucleotide synthesis, a practical approach”, Ed. M. J.Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A PracticalApproach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1,Modern machine-aided methods of oligodeoxyribonucleotide synthesis,Chapter. 2, Oligoribonucleotide synthesis, Chapter3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein. Modification described inWO 00/44895, WO01/75164, or WO02/44321 can be used herein. Thedisclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.Tetrahedron Len. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,19%, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No.5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Nucleobases References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191:5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.

Linkers

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, Alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,allylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R¹)₂,C(O), cleavable linking group, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclic; where R¹ is hydrogen, acyl, aliphatic or substitutedaliphatic.

In one embodiment, the linker is—[(P-Q-R)_(q)—X—(P′-Q′-R′)_(q)′]_(q)-T-, wherein:

P, R, T, P′, R′ and T are each independently for each occurrence absent,CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, CH═N—O,

or heterocyclyl;

Q and Q′ are each independently for each occurrence absent, —(CH₂)_(n)—,—C(R¹)(R²)(CH₂)_(n)—, —(CH₂)_(a)C(R¹)(R²)—, —(CH₂CH₂O)_(n)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is absent or a cleavable linking group;

R^(a) is H or an amino acid side chain;

R¹ and R² are each independently for each occurrence H, CH₃, OH, SH orN(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q, q′ and q″ are each independently for each occurrence 0-20 and whereinthe repeating unit can be the same or different;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup.

In certain embodiments, the linker is a branched linker. The branchpointof the branched linker may be at least trivalent, but may be atetravalent, pentavalent or hexavalent atom, or a group presenting suchmultiple valencies. In certain embodiments, the branchpoint is, —N,—N(O)—C, —O—C, —S—C, —SS—C, —C(O)N(O)—C, —OC(O)N(O)—C, —N(O)C(O)—C, or—N(O)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. Ina cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynylene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R″ and R^(B) are the R groups of thetwo adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Ligands

A wide variety of entities can be coupled to the oligonucleotides andlipids of the present invention. Preferred moieties are ligands, whichare coupled, preferably covalently, either directly or indirectly via anintervening tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Ligands providing enhancedaffinity for a selected target are also termed targeting ligands.Preferred ligands for conjugation to the lipids of the present inventionare targeting ligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In certain embodiments, the endosomolytic ligand assumesits active conformation at endosomal pH. The “active” conformation isthat conformation in which the endosomolytic ligand promotes lysis ofthe endosome and/or transport of the composition of the invention, orits components, from the endosome to the cytoplasm of the cell.Exemplary endosomolytic ligands include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of peptide based endosomolyticligands are shown in Table 5.

TABLE 5  List of peptides with endosomolytic activity. NameSequence (N to C) Ref. GALA AALEALAEALEALAEALEALAEAAAAGGC 1 EALAAALAEALAEALAEALAEALAEALAAAAGGC 2 ALEALAEALEALAEA 3 INF-7GLFEAIEGFIENGWEGMIWDYG 4 Inf HA-2 GLFGAIAGFIENGWEGMIDGWYG 5 diINF-7GLF EAI EGFI ENGW EGMI DGWYGC 5 GLF EAI EGFI ENGW EGMI DGWYGC diINF3GLF EAI EGFI ENGW EGMI DGGC 6 GLF EAI EGFI ENGW EGMI DGGC GLFGLFGALAEALAEALAEHLAEALAEALEALAAGGSC 6 GALA-INF3GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6 INF-5 GLF EAI EGFI ENGW EGnI DG K 4GLF EAI EGFI ENGW EGnI DG n, norleucine References 1. Subbarao et al.,Biochemistry, 1987, 26: 2964-2972. 2. Vogel et al., J. Am. Chem. Soc.,1996, 118: 1581-1586 3. Turk. M. J., Reddy, J. A. et al. (2002).Characterization of a novel pH-sensitive peptide that enhances drugrelease from folate-targeted liposomes at endosomal pHs. Biochim.Biophys. Acta 1559, 56-68. 4. Plank, C. Oberhauser, B. Mechtler, K.Koch, C. Wagner, E. (1994). The influence of endosome-disruptivepeptides on gene transfer using synthetic virus-like gene transfersystems, J. Biol. Chem. 269 12918-12924. 5. Mastrobattista, E., Koning,G. A. et al. (2002). Functional characterization of anendosome-disruptive peptide and its application in cytosolic delivery ofimmunoliposome-entrapped proteins. J. Biol. Chem. 277, 27135-43. 6.Oberhauser, B., Plank, C. et al. (1995). Enhancing endosomal exit ofnucleic acids using pH-sensitive viral fusion peptides. Deliv.Strategies Antisense Oligonucleotide Ther. 247-66.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropylmethacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 6 shows some examples of targetingligands and their associated receptors.

TABLE 6 Targeting Ligands and their associated receptors Liver CellsLigand Receptor 1) Parenchymal Galactose ASGP-R Cell (PC)(Asiologlycoprotein (Hepatocytes) receptor) Gal NAc ASPG-R(n-acetyl-galactosamine) Gal NAc Receptor Lactose Asialofetuin ASPG-r 2)Sinusoidal Hyaluronan Hyaluronan receptor Endothelial ProcollagenProcollagen receptor Cell (SEC) Negatively charged molecules Scavengerreceptors Mannose Mannose receptors N-acetyl Glucosamine Scavengerreceptors Immunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptormediated transcytosis Transferrin Receptor mediated transcytosisAlbumins Non-specific Sugar-Albumin conjugates Mannose-6-phosphateMannose-6-phosphate receptor 3) Kupffer Cell Mannose Mannose receptors(KC) Fucose Fucose receptors Albumins Non-specific Mannose-albuminconjugates

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL):

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g.; about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long (see Table 7, for example).

TABLE 7  Exemplary Cell Permeation Peptides. PR-39RRRPRPPYLPRPRPPPFFPPRLPPRIPPGF PPRFPPRFPGKR-NH2 IndolicidinILPWKWPWWPWRR-NH2 Amino acid Sequence Reference PenetratinRQIKIWFQNRRMKWKK Derossi et al., J. Biol. Chem. 269:10444, 1994Tat fragment GRKKRRQRRRPPQC Vives et al., J. Biol. (48-60)Chem., 272:16010, 1997 Signal Sequence- GALFLGWLGAAGSTMGAWSQPKKKRChaloin et al., Biochem. based peptide KV Biophys. Res. Commun.,243:601, 1998 PVEC LLIILRRRIRKQAHAHSK Elmquist et al., Exp. CellRes., 269:237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKILPooga et al., FASEB J., 12:67, 1998 Amphiphilic KLALKLALKALKAALKLAOehlke et al., Mol. Ther., model peptide 2:339, 2000 Arg₉ RRRRRRRRRMitchell et al., J. Pept. Res., 56:318, 2000 Bacterial cell KFFKFFKFFKwall permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFL RNLVPRTES Cecropin P1SWLSKTAKKLENSAKKRISEGIAIAIQG GPR α-defensin ACYCRIPACIAGERRYGTCIYQGRLWAFCC b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTC YRGKAKCCK BactenecinRKCRIVVIRVCR

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Tip or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequenceAALLPVLLAAP) containing a hydrophobic MTS can also be a targetingmoiety. The peptide moiety can be a “delivery” peptide, which can carrylarge polar molecules including peptides, oligonucleotides, and proteinacross cell membranes. For example, sequences from the HIV Tat protein(GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein(RQIKIWFQNRRMKWKK) have been found to be capable of functioning asdelivery peptides: A peptide or peptidomimetic can be encoded by arandom sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide orpeptidomimetic tethered to an iRNA agent via an incorporated monomerunit is a cell targeting peptide such as an arginine-glycine-asparticacid (RGD)-peptide, or RGD mimic. A peptide moiety can range in lengthfrom about 5 amino acids to about 40 amino acids. The peptide moietiescan have a structural modification, such as to increase stability ordirect conformational properties. Any of the structural modificationsdescribed below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner aal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an αvβ3 integrin. Thus,one could use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the αvβ3 integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogenesis. Preferred conjugates of this type ligands that targetsPECAM-1, VEGF, or other cancer gene, e.g., a cancer gene describedherein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an iRNA agent and/orthe carrier oligomer can be an amphipathic α-helical peptide. Exemplaryamphipathic α-helical peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide(BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfishintestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides,esculentinis-1, and caerins. A number of factors will preferably beconsidered to maintain the integrity of helix stability. For example, amaximum number of helix stabilization residues will be utilized (e.g.,leu, ala, or lys), and a minimum number helix destabilization residueswill be utilized (e.g., proline, or cyclic monomeric units. The cappingresidue will be considered (for example Gly is an exemplary N-cappingresidue and/or C-terminal amidation can be used to provide an extraH-bond to stabilize the helix. Formation of salt bridges betweenresidues with opposite charges, separated by i±3, or i±4 positions canprovide stability. For example, cationic residues such as lysine,arginine, homo-arginine, ornithine or histidine can form salt bridgeswith the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an aptamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Exemplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligands amenable to the invention are described in copendingapplications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether. The ligand or tethered ligand may be present on amonomer when said monomer is incorporated into the growing strand. Insome embodiments, the ligand may be incorporated via coupling to a“precursor” monomer after said “precursor” monomer has been incorporatedinto the growing strand. For example, a monomer having, e.g., anamino-terminated tether (i.e., having no associated ligand), e.g.,TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer into the strand, a ligand having an electrophilicgroup, e.g., a pentafluorophenyl ester or aldehyde group, cansubsequently be attached to the precursor monomer by coupling theelectrophilic group of the ligand with the terminal nucleophilic groupof the precursor monomer's tether.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662;5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434;6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631;6,559,279; each of which is herein incorporated by reference.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide including a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotideincluding inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencesincluding such replacement moieties are embodiments of the invention.

By “Factor VII” as used herein is meant a Factor VII mRNA, protein,peptide, or polypeptide. The term “Factor VII” is also known in the artas AII32620, Cf7, Coagulation factor VII precursor, coagulation factorVII, FVII, Serum prothrombin conversion accelerator, FVII coagulationprotein, and eptacog alfa.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, the term “strand including a sequence” refers to anoligonucleotide including a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used in the context of a nucleotide pair, means aclassic Watson-Crick pair, i.e., GC, AT, or AU. It also extends toclassic Watson-Crick pairings where one or both of the nucleotides hasbeen modified as described herein, e.g., by a those modification or aphosphate backpone modification. It can also include pairing with aninosine or other entity that does not substantially alter the basepairing properties.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide including the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide including thesecond nucleotide sequence, as will be understood by the skilled person.Complementarity can include, full complementarity, substantialcomplementarity, and sufficient complementarity to allow hybridizationunder physiological conditions, e.g, under physiologically relevantconditions as may be encountered inside an organism. Fullcomplementarity refers to complementarity, as defined above for anindividual pair, at all of the pairs of the first and second sequence.When a sequence is “substantially complementary” with respect to asecond sequence herein, the two sequences can be fully complementary, orthey may form one or more, but generally not more than 4, 3 or 2mismatched base pairs upon hybridization, while retaining the ability tohybridize under the conditions most relevant to their ultimateapplication. Substantial complementarity can also be defined ashybridization under stringent conditions, where stringent conditions mayinclude: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C.for 12-16 hours followed by washing. The skilled person will be able todetermine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA including one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide includes a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposes of theinvention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary”, “substantiallycomplementary” and sufficient complementarity to allow hybridizationunder physiological conditions, e.g, under physiologically relevantconditions as may be encountered inside an organism, may be usedhereinwith respect to the base matching between the sense strand and theantisense strand of a dsRNA, or between the antisense strand of a dsRNAand a target sequence, as will be understood from the context of theiruse.

As used herein, a polynucleotide which is “complementary, e.g.,substantially complementary to at least part of” a messenger RNA (mRNA)refers to a polynucleotide which is complementary, e.g., substantiallycomplementary, to a contiguous portion of the mRNA of interest (e.g.,encoding Factor VII). For example, a polynucleotide is complementary toat least a part of a Factor VII mRNA if the sequence is substantiallycomplementary to a non-interrupted portion of an mRNA encoding FactorVII.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aribonucleic acid molecule, or complex of ribonucleic acid molecules,having a duplex structure including two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker.” The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. In addition to the duplex structure, a dsRNA may compriseone or more nucleotide overhangs. A dsRNA as used herein is alsoreferred to as a “small inhibitory RNA,” “siRNA,” “siRNA agent,” “iRNAagent” or “RNAi agent.”

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

The term “identity” is the relationship between two or morepolynucleotide sequences, as determined by comparing the sequences.Identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match between strings ofsuch sequences. While there exist a number of methods to measureidentity between two polynucleotide sequences, the term is well known toskilled artisans (see, e.g., Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press (1987); and Sequence Analysis Primer,Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York(1991)). “Substantially identical,” as used herein, means there is avery high degree of homology (preferably 100% sequence identity) betweenthe sense strand of the dsRNA and the corresponding part of the targetgene. However, dsRNA having greater than 90%, or 95% sequence identitymay be used in the invention, and thus sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence can be tolerated. Although 100% identity is preferred, thedsRNA may contain single or multiple base-pair random mismatches betweenthe RNA and the target gene.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of,” in as far as theyrefer to the Factor VII gene, herein refer to the at least partialsuppression of the expression of the Factor VII gene, as manifested by areduction of the amount of mRNA from the Factor VII gene which may beisolated from a first cell or group of cells in which the Factor VIIgene is transcribed and which has or have been treated such that theexpression of the Factor VII gene is inhibited, as compared to a secondcell or group of cells substantially identical to the first cell orgroup of cells but which has or have not been so treated (controlcells). The degree of inhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Factor VII genetranscription, e.g. the amount of protein encoded by the Factor VII genewhich is secreted by a cell, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, Factor VII gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given siRNA inhibitsthe expression of the Factor VII gene by a certain degree and thereforeis encompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of the Factor VII gene issuppressed by at least about 20%, 25%, 35%, 40% or 50% by administrationof the double-stranded oligonucleotide of the invention. In oneembodiment, the Factor VII gene is suppressed by at least about 60%,70%, or 80% by administration of the double-stranded oligonucleotide ofthe invention. In a more preferred embodiment, the Factor VII gene issuppressed by at least about 85%, 90%, or 95% by administration of thedouble-stranded oligonucleotide of the invention.

The terms “treat,” “treatment,” and the like, refer to relief from oralleviation of a disease or disorder. In the context of the inventioninsofar as it relates to any of the other conditions recited hereinbelow (e.g., a Factor VII-mediated condition other than a thromboticdisorder), the terms “treat,” “treatment,” and the like mean to relieveor alleviate at least one symptom associated with such condition, or toslow or reverse the progression of such condition.

A “therapeutically relevant” composition can alleviate a disease ordisorder, or a symptom of a disease or disorder when administered at anappropriate dose.

As used herein, the term “Factor VII-mediated condition or disease” andrelated terms and phrases refer to a condition or disorder characterizedby inappropriate, e.g., greater than normal, Factor VII activity.Inappropriate Factor VII functional activity might arise as the resultof Factor VII expression in cells which normally do not express FactorVII, or increased Factor VII expression (leading to, e.g., a symptom ofa viral hemorrhagic fever, or a thrombus). A Factor VII-mediatedcondition or disease may be completely or partially mediated byinappropriate Factor VII functional activity. However, a FactorVII-mediated condition or disease is one in which modulation of FactorVII results in some effect on the underlying condition or disorder(e.g., a Factor VII inhibitor results in some improvement in patientwell-being in at least some patients).

A “hemorrhagic fever” includes a combination of illnesses caused by aviral infection. Fever and gastrointestinal symptoms are typicallyfollowed by capillary hemorrhaging.

A “coagulopathy” is any defect in the blood clotting mechanism of asubject.

As used herein, a “thrombotic disorder” is any disorder, preferablyresulting from unwanted FVII expression, including any disordercharacterized by unwanted blood coagulation.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of aviral hemorrhagic fever, or an overt symptom of such disorder, e.g.,hemorrhaging, fever, weakness, muscle pain, headache, inflammation, orcirculatory shock. The specific amount that is therapeutically effectivecan be readily determined by ordinary medical practitioner, and may varydepending on factors known in the art, such as, e.g. the type ofthrombotic disorder, the patient's history and age, the stage of thedisease, and the administration of other agents.

As used herein, a “pharmaceutical composition” includes apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

Characteristic of Nucleic Acid-Lipid Particles

In certain embodiments, the invention relates to methods andcompositions for producing lipid-encapsulated nucleic acid particles inwhich nucleic acids are encapsulated within a lipid layer. Such nucleicacid-lipid particles, incorporating siRNA oligonucleotides, arecharacterized using a variety of biophysical parameters including: (1)drug to lipid ratio; (2) encapsulation efficiency; and (3) particlesize. High drug to lipid rations, high encapsulation efficiency, goodnuclease resistance and serum stability and controllable particle size,generally less than 200 nm in diameter are desirable. In addition, thenature of the nucleic acid polymer is of significance, since themodification of nucleic acids in an effort to impart nuclease resistanceadds to the cost of therapeutics while in many cases providing onlylimited resistance. Unless stated otherwise, these criteria arecalculated in this specification as follows:

Nucleic acid to lipid ratio is the amount of nucleic acid in a definedvolume of preparation divided by the amount of lipid in the same volume.This may be on a mole per mole basis or on a weight per weight basis, oron a weight per mole basis. For final, administration-readyformulations, the nucleic acid:lipid ratio is calculated after dialysis,chromatography and/or enzyme (e.g., nuclease) digestion has beenemployed to remove as much of the external nucleic acid as possible;

Encapsulation efficiency refers to the drug to lipid ratio of thestarting mixture divided by the drug to lipid ratio of the final,administration competent formulation. This is a measure of relativeefficiency. For a measure of absolute efficiency, the total amount ofnucleic acid added to the starting mixture that ends up in theadministration competent formulation, can also be calculated. The amountof lipid lost during the formulation process may also be calculated.Efficiency is a measure of the wastage and expense of the formulation;and

Size indicates the size (diameter) of the particles formed. Sizedistribution may be determined using quasi-elastic light scattering(QELS) on a Nicomp Model 370 sub-micron particle sizer. Particles under200 nm are preferred for distribution to neo-vascularized (leaky)tissues, such as neoplasms and sites of inflammation.

Methods of Preparing Lipid Particles

The methods and compositions of the invention make use of certaincationic lipids, the synthesis, preparation and characterization ofwhich is described below and in the accompanying Examples. In addition,the present invention provides methods of preparing lipid particles,including those associated with a therapeutic agent, e.g., a nucleicacid. In the methods described herein, a mixture of lipids is combinedwith a buffered aqueous solution of nucleic acid to produce anintermediate mixture containing nucleic acid encapsulated in lipidparticles wherein the encapsulated nucleic acids are present in anucleic acid/lipid ratio of about 3 wt % to about 25 wt %, preferably 5to 15 wt %. The intermediate mixture may optionally be sized to obtainlipid-encapsulated nucleic acid particles wherein the lipid portions areunilamellar vesicles, preferably having a diameter of 30 to 150 nm, morepreferably about 40 to 90 nm. The pH is then raised to neutralize atleast a portion of the surface charges on the lipid-nucleic acidparticles, thus providing an at least partially surface-neutralizedlipid-encapsulated nucleic acid composition.

As described above, several of these cationic lipids are amino lipidsthat are charged at a pH below the pK_(a) of the amino group andsubstantially neutral at a pH above the pK_(a). These cationic lipidsare termed titratable cationic lipids and can be used in theformulations of the invention using a two-step process. First, lipidvesicles can be formed at the lower pH with titratable cationic lipidsand other vesicle components in the presence of nucleic acids. In thismanner, the vesicles will encapsulate and entrap the nucleic acids.Second, the surface charge of the newly formed vesicles can beneutralized by increasing the pH of the medium to a level above thepK_(a) of the titratable cationic lipids present, i.e., to physiologicalpH or higher. Particularly advantageous aspects of this process includeboth the facile removal of any surface adsorbed nucleic acid and aresultant nucleic acid delivery vehicle which has a neutral surface.Liposomes or lipid particles having a neutral surface are expected toavoid rapid clearance from circulation and to avoid certain toxicitieswhich are associated with cationic liposome preparations. Additionaldetails concerning these uses of such titratable cationic lipids in theformulation of nucleic acid-lipid particles are provided in U.S. Pat.No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein byreference.

It is further noted that the vesicles formed in this manner provideformulations of uniform vesicle size with high content of nucleic acids.Additionally, the vesicles have a size range of from about 30 to about150 nm, more preferably about 30 to about 90 nm.

Without intending to be bound by any particular theory, it is believedthat the very high efficiency of nucleic acid encapsulation is a resultof electrostatic interaction at low pH. At acidic pH (e.g. pH 4.0) thevesicle surface is charged and binds a portion of the nucleic acidsthrough electrostatic interactions. When the external acidic buffer isexchanged for a more neutral buffer (e.g. pH 7.5) the surface of thelipid particle or liposome is neutralized, allowing any external nucleicacid to be removed. More detailed information on the formulation processis provided in various publications (e.g., U.S. Pat. No. 6,287,591 andU.S. Pat. No. 6,858,225).

In view of the above, the present invention provides methods ofpreparing lipid/nucleic acid formulations. In the methods describedherein, a mixture of lipids is combined with a buffered aqueous solutionof nucleic acid to produce an intermediate mixture containing nucleicacid encapsulated in lipid particles, e.g., wherein the encapsulatednucleic acids are present in a nucleic acid/lipid ratio of about 10 wt %to about 20 wt %. The intermediate mixture may optionally be sized toobtain lipid-encapsulated nucleic acid particles wherein the lipidportions are unilamellar vesicles, preferably having a diameter of 30 to150 nm, more preferably about 40 to 90 nm. The pH is then raised toneutralize at least a portion of the surface charges on thelipid-nucleic acid particles, thus providing an at least partiallysurface-neutralized lipid-encapsulated nucleic acid composition.

In certain embodiments, the mixture of lipids includes at least twolipid components: a first amino lipid component of the present inventionthat is selected from among lipids which have a pKa such that the lipidis cationic at pH below the pKa and neutral at pH above the pKa, and asecond lipid component that is selected from among lipids that preventparticle aggregation during lipid-nucleic acid particle formation. Inparticular embodiments, the amino lipid is a novel cationic lipid of thepresent invention.

In preparing the nucleic acid-lipid particles of the invention, themixture of lipids is typically a solution of lipids in an organicsolvent. This mixture of lipids can then be dried to form a thin film orlyophilized to form a powder before being hydrated with an aqueousbuffer to form liposomes. Alternatively, in a preferred method, thelipid mixture can be solubilized in a water miscible alcohol, such asethanol, and this ethanolic solution added to an aqueous bufferresulting in spontaneous liposome formation. In most embodiments, thealcohol is used in the form in which it is commercially available. Forexample, ethanol can be used as absolute ethanol (100%), or as 95%ethanol, the remainder being water. This method is described in moredetail in U.S. Pat. No. 5,976,567).

In one exemplary embodiment, the mixture of lipids is a mixture ofcationic lipids, neutral lipids (other than a cationic lipid), a sterol(e.g., cholesterol) and a PEG-modified lipid (e.g., a PEG-DMG orPEG-cDMA) in an alcohol solvent. In preferred embodiments, the lipidmixture consists essentially of a cationic lipid, a neutral lipid,cholesterol and a PEG-modified lipid in alcohol, more preferablyethanol. In further preferred embodiments, the first solution consistsof the above lipid mixture in molar ratios of about 20-70% cationiclipid:5-45% neutral lipid:20-55% cholesterol:0.5-15% PEG-modified lipid.In still further preferred embodiments, the first solution consistsessentially of a lipid chosen from Table 1, DSPC, Chol and PEG-DMG orPEG-cDMA, more preferably in a molar ratio of about 20-60% cationiclipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA. In particularembodiments, the molar lipid ratio is approximately 50/10/38.5/1.5 (mol% cationic lipid/DSPC/Chol/PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1/34.3/1.4 (mol % cationic lipid/DPPC/Chol/PEG-cDMA), 40/15/40/5(mol % cationic lipid/DSPC/Chol/PEG-DMG), 50/10/35/4.5/0.5 (mol %cationic lipid/DSPC/Chol/PEG-DSG or GalNAc3-PEG-DSG), 50/10/35/5(cationic lipid/DSPC/Chol/PEG-DMG), 40/10/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-cDMA). In another group of preferredembodiments, the neutral lipid in these compositions is replaced withPOPC, DPPC, DOPE or SM. In accordance with the invention, the lipidmixture is combined with a buffered aqueous solution that may containthe nucleic acids. The buffered aqueous solution of is typically asolution in which the buffer has a pH of less than the pK_(a) of theprotonatable lipid in the lipid mixture. Examples of suitable buffersinclude citrate, phosphate, acetate, and MES. A particularly preferredbuffer is citrate buffer. Preferred buffers will be in the range of1-1000 mM of the anion, depending on the chemistry of the nucleic acidbeing encapsulated, and optimization of buffer concentration may besignificant to achieving high loading levels (see, e.g., U.S. Pat. No.6,287,591 and U.S. Pat. No. 6,858,225). Alternatively, pure wateracidified to pH 5-6 with chloride, sulfate or the like may be useful. Inthis case, it may be suitable to add 5% glucose, or another non-ionicsolute which will balance the osmotic potential across the particlemembrane when the particles are dialyzed to remove ethanol, increase thepH, or mixed with a pharmaceutically acceptable carrier such as normalsaline. The amount of nucleic acid in buffer can vary, but willtypically be from about 0.01 mg/mL to about 200 mg/mL, more preferablyfrom about 0.5 mg/mL to about 50 mg/mL.

The mixture of lipids and the buffered aqueous solution of therapeuticnucleic acids is combined to provide an intermediate mixture. Theintermediate mixture is typically a mixture of lipid particles havingencapsulated nucleic acids. Additionally, the intermediate mixture mayalso contain some portion of nucleic acids which are attached to thesurface of the lipid particles (liposomes or lipid vesicles) due to theionic attraction of the negatively-charged nucleic acids andpositively-charged lipids on the lipid particle surface (the aminolipids or other lipid making up the protonatable first lipid componentare positively charged in a buffer having a pH of less than the pK_(a)of the protonatable group on the lipid). In one group of preferredembodiments, the mixture of lipids is an alcohol solution of lipids andthe volumes of each of the solutions is adjusted so that uponcombination, the resulting alcohol content is from about 20% by volumeto about 45% by volume. The method of combining the mixtures can includeany of a variety of processes, often depending upon the scale offormulation produced. For example, when the total volume is about 10-20mL or less, the solutions can be combined in a test tube and stirredtogether using a vortex mixer. Large-scale processes can be carried outin suitable production scale glassware.

Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleicacid) complexes which are produced by combining the lipid mixture andthe buffered aqueous solution of therapeutic agents (nucleic acids) canbe sized to achieve a desired size range and relatively narrowdistribution of lipid particle sizes. Preferably, the compositionsprovided herein will be sized to a mean diameter of from about 70 toabout 200 nm, more preferably about 90 to about 130 nm. Severaltechniques are available for sizing liposomes to a desired size. Onesizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles (SUVs) less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize determination. For certain methods herein, extrusion is used toobtain a uniform vesicle size.

Extrusion of liposome compositions through a small-pore polycarbonatemembrane or an asymmetric ceramic membrane results in a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing.

In particular embodiments, methods of the present invention furthercomprise a step of neutralizing at least some of the surface charges onthe lipid portions of the lipid-nucleic acid compositions. By at leastpartially neutralizing the surface charges, unencapsulated nucleic acidis freed from the lipid particle surface and can be removed from thecomposition using conventional techniques. Preferably, unencapsulatedand surface adsorbed nucleic acids are removed from the resultingcompositions through exchange of buffer solutions. For example,replacement of a citrate buffer (pH about 4.0, used for forming thecompositions) with a HEPES-buffered saline (FIBS pH about 7.5) solution,results in the neutralization of liposome surface and nucleic acidrelease from the surface. The released nucleic acid can then be removedvia chromatography using standard methods, and then switched into abuffer with a pH above the pKa of the lipid used.

Optionally the lipid vesicles (i.e., lipid particles) can be formed byhydration in an aqueous buffer and sized using any of the methodsdescribed above prior to addition of the nucleic acid. As describedabove, the aqueous buffer should be of a pH below the pKa of the aminolipid. A solution of the nucleic acids can then be added to these sized,preformed vesicles. To allow encapsulation of nucleic acids into such“pre-formed” vesicles the mixture should contain an alcohol, such asethanol. In the case of ethanol, it should be present at a concentrationof about 20% (w/w) to about 45% (w/w). In addition, it may be necessaryto warm the mixture of pre-formed vesicles and nucleic acid in theaqueous buffer-ethanol mixture to a temperature of about 25° C. to about50° C. depending on the composition of the lipid vesicles and the natureof the nucleic acid. It will be apparent to one of ordinary skill in theart that optimization of the encapsulation process to achieve a desiredlevel of nucleic acid in the lipid vesicles will require manipulation ofvariable such as ethanol concentration and temperature. Examples ofsuitable conditions for nucleic acid encapsulation are provided in theExamples. Once the nucleic acids are encapsulated within the preformedvesicles, the external pH can be increased to at least partiallyneutralize the surface charge. Unencapsulated and surface adsorbednucleic acids can then be removed as described above.

Method of Use

The lipid particles of the invention may be used to deliver atherapeutic agent to a cell, in vitro or in vivo. In particularembodiments, the therapeutic agent is a nucleic acid, which is deliveredto a cell using a nucleic acid-lipid particles of the invention. Whilethe following description of various methods of using the lipidparticles and related pharmaceutical compositions of the invention areexemplified by description related to nucleic acid-lipid particles, itis understood that these methods and compositions may be readily adaptedfor the delivery of any therapeutic agent for the treatment of anydisease or disorder that would benefit from such treatment.

In certain embodiments, the invention provides methods for introducing anucleic acid into a cell. Preferred nucleic acids for introduction intocells are siRNA, immune-stimulating oligonucleotides, plasmids,antisense and ribozymes. These methods may be carried out by contactingthe particles or compositions of the invention with the cells for aperiod of time sufficient for intracellular delivery to occur.

The compositions of the invention can be adsorbed to almost any celltype, e.g., tumor cell lines, including but not limited to HeLa, HCT116,A375, MCF7, B16F10, Hep3b, HUH7, HepG2, Skov3, U87, and PC3 cell lines.Once adsorbed, the nucleic acid-lipid particles can either beendocytosed by a portion of the cells, exchange lipids with cellmembranes, or fuse with the cells. Transfer or incorporation of thenucleic acid portion of the complex can take place via any one of thesepathways. Without intending to be limited with respect to the scope ofthe invention, it is believed that in the case of particles taken upinto the cell by endocytosis the particles then interact with theendosomal membrane, resulting in destabilization of the endosomalmembrane, possibly by the formation of non-bilayer phases, resulting inintroduction of the encapsulated nucleic acid into the cell cytoplasm.Similarly in the case of direct fusion of the particles with the cellplasma membrane, when fusion takes place, the liposome membrane isintegrated into the cell membrane and the contents of the liposomecombine with the intracellular fluid. Contact between the cells and thelipid-nucleic acid compositions, when carried out in vitro, will takeplace in a biologically compatible medium. The concentration ofcompositions can vary widely depending on the particular application,but is generally between about 1 mmol and about 10 mmol. In certainembodiments, treatment of the cells with the lipid-nucleic acidcompositions will generally be carried out at physiological temperatures(about 37° C.) for periods of time from about 1 to 24 hours, preferablyfrom about 2 to 8 hours. For in vitro applications, the delivery ofnucleic acids can be to any cell grown in culture, whether of plant oranimal origin, vertebrate or invertebrate, and of any tissue or type. Inpreferred embodiments, the cells will be animal cells, more preferablymammalian cells, and most preferably human cells.

In one group of embodiments, a lipid-nucleic acid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/mL, more preferably about 2×10⁴ cells/mL.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.

Typical applications include using well known procedures to provideintracellular delivery of siRNA to knock down or silence specificcellular targets. Alternatively applications include delivery of DNA ormRNA sequences that code for therapeutically useful polypeptides. Inthis manner, therapy is provided for genetic diseases by supplyingdeficient or absent gene products (i.e., for Duchenne's dystrophy, seeKunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cysticfibrosis, see Goodfellow, Nature 341:102-103 (1989)). Other uses for thecompositions of the invention include introduction of antisenseoligonucleotides in cells (see, Bennett, et al., Mol. Pharm.41:1023-1033 (1992)).

Alternatively, the compositions of the invention can also be used fordeliver of nucleic acids to cells in vivo, using methods which are knownto those of skill in the art. With respect to application of theinvention for delivery of DNA or mRNA sequences, Zhu, et al., Science261:209-211 (1993), incorporated herein by reference, describes theintravenous delivery of cytomegalovirus (CMV)-chloramphenicolacetyltransferase (CAT) expression plasmid using DOTMA-DOPE complexes.Hyde, et al., Nature 362:250-256 (1993), incorporated herein byreference, describes the delivery of the cystic fibrosis transmembraneconductance regulator (CFTR) gene to epithelia of the airway and toalveoli in the lung of mice, using liposomes. Brigham, et al., Am. J.Med. Sci. 298:278-281 (1989), incorporated herein by reference,describes the in vivo transfection of lungs of mice with a functioningprokaryotic gene encoding the intracellular enzyme, chloramphenicolacetyltransferase (CAT). Thus, the compositions of the invention can beused in the treatment of infectious diseases.

Therefore, in another aspect, the formulations of the invention can beused to silence or modulate a target gene such as but not limited toFVII, Eg5; PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene,Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene,Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene,Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene,survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase IIalpha gene, p73 gene, p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene,RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, tumorsuppressor genes, p53 tumor suppressor gene, p53 family member DN-p63,pRb tumor suppressor gene, APC1 tumor suppressor gene, BRCA1 tumorsuppressor gene, PTEN tumor suppressor gene, mLL fusion gene, BCR/ABLfusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusion gene, TLS/FUS1 fusiongene, PAX3/FKHR fusion gene, AML1/ETO fusion gene, alpha v-integringene, Flt-1 receptor gene, tubulin gene, Human Papilloma Virus gene, agene required for Human Papilloma Virus replication, HumanImmunodeficiency Virus gene, a gene required for Human ImmunodeficiencyVirus replication, Hepatitis A Virus gene, a gene required for HepatitisA Virus replication, Hepatitis B Virus gene, a gene required forHepatitis B Virus replication, Hepatitis C Virus gene, a gene requiredfor Hepatitis C Virus replication, Hepatitis D Virus gene, a generequired for Hepatitis D Virus replication, Hepatitis E Virus gene, agene required for Hepatitis E Virus replication, Hepatitis F Virus gene,a gene required for Hepatitis F Virus replication, Hepatitis G Virusgene, a gene required for Hepatitis G Virus replication, Hepatitis HVirus gene, a gene required for Hepatitis H Virus replication,Respiratory Syncytial Virus gene, a gene that is required forRespiratory Syncytial Virus replication, Herpes Simplex Virus gene, agene that is required for Herpes Simplex Virus replication, herpes.Cytomegalovirus gene, a gene that is required for herpes Cytomegalovirusreplication, herpes Epstein Barr Virus gene, a gene that is required forherpes Epstein Barr Virus replication, Kaposi's Sarcoma-associatedHerpes Virus gene, a gene that is required for Kaposi'sSarcoma-associated Herpes Virus replication, JC Virus gene, human genethat is required for JC Virus replication, myxovirus gene, a gene thatis required for myxovirus gene replication, rhinovirus gene, a gene thatis required for rhinovirus replication, coronavirus gene, a gene that isrequired for coronavirus replication, West Nile Virus gene, a gene thatis required for West Nile Virus replication, St. Louis Encephalitisgene, a gene that is required for St. Louis Encephalitis replication,Tick-borne encephalitis virus gene, a gene that is required forTick-borne encephalitis virus replication, Murray Valley encephalitisvirus gene, a gene that is required for Murray Valley encephalitis virusreplication, dengue virus gene, a gene that is required for dengue virusgene replication, Simian Virus 40 gene, a gene that is required forSimian Virus 40 replication, Human T Cell Lymphotropic Virus gene, agene that is required for Human T Cell Lymphotropic Virus replication,Moloney-Murine Leukemia Virus gene, a gene that is required forMoloney-Murine Leukemia Virus replication, encephalomyocarditis virusgene, a gene that is required for encephalomyocarditis virusreplication, measles virus gene, a gene that is required for measlesvirus replication, Vericella zoster virus gene, a gene that is requiredfor Vericella zoster virus replication, adenovirus gene, a gene that isrequired for adenovirus replication, yellow fever virus gene, a genethat is required for yellow fever virus replication, poliovirus gene, agene that is required for poliovirus replication, poxvirus gene, a genethat is required for poxvirus replication, plasmodium gene, a gene thatis required for plasmodium gene replication, Mycobacterium ulceransgene, a gene that is required for Mycobacterium ulcerans replication,Mycobacterium tuberculosis gene, a gene that is required forMycobacterium tuberculosis replication, Mycobacterium leprae gene, agene that is required for Mycobacterium leprae replication,Staphylococcus aureus gene, a gene that is required for Staphylococcusaureus replication, Streptococcus pneumoniae gene, a gene that isrequired for Streptococcus pneumoniae replication, Streptococcuspyogenes gene, a gene that is required for Streptococcus pyogenesreplication, Chlamydia pneumoniae gene, a gene that is required forChlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly. Inparticular embodiments, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection. For oneexample, see Stadler, et U.S. Pat. No. 5,286,634, which is incorporatedherein by reference. Intracellular nucleic acid delivery has also beendiscussed in Straubringer, et al., METHODS IN ENZYMOLOGY, AcademicPress, New York. 101:512-527 (1983); Mannino, et al., Biotechniques6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. Drug Carrier Syst.6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993). Stillother methods of administering lipid-based therapeutics are describedin, for example, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S.Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871;Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No.4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

In other methods, the pharmaceutical preparations may be contacted withthe target tissue by direct application of the preparation to thetissue. The application may be made by topical, “open” or “closed”procedures. By “topical,” it is meant the direct application of thepharmaceutical preparation to a tissue exposed to the environment, suchas the skin, oropharynx, external auditory canal, and the like. “Open”procedures are those procedures which include incising the skin of apatient and directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue. “Closed” procedures are invasiveprocedures in which the internal target tissues are not directlyvisualized, but accessed via inserting instruments through small woundsin the skin. For example, the preparations may be administered to theperitoneum by needle lavage. Likewise, the pharmaceutical preparationsmay be administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrazamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

The lipid-nucleic acid compositions can also be administered in anaerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci.298(4):278-281 (1989)) or by direct injection at the site of disease(Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, NewYork. pp. 70-71 (1994)).

The methods of the invention may be practiced in a variety of hosts.Preferred hosts include mammalian species, such as humans, non-humanprimates, dogs, cats, cattle, horses, sheep, and the like.

Dosages for the lipid-therapeutic agent particles of the invention willdepend on the ratio of therapeutic agent to lipid and the administratingphysician's opinion based on age, weight, and condition of the patient.

In one embodiment, the invention provides a method of modulating theexpression of a target polynucleotide or polypeptide. These methodsgenerally comprise contacting a cell with a lipid particle of theinvention that is associated with a nucleic acid capable of modulatingthe expression of a target polynucleotide or polypeptide. As usedherein, the term “modulating” refers to altering the expression of atarget polynucleotide or polypeptide. In different embodiments,modulating can mean increasing or enhancing, or it can mean decreasingor reducing. Methods of measuring the level of expression of a targetpolynucleotide or polypeptide are known and available in the arts andinclude, e.g., methods employing reverse transcription-polymerase chainreaction (RT-PCR) and immunohistochemical techniques. In particularembodiments, the level of expression of a target polynucleotide orpolypeptide is increased or reduced by at least 10%, 20%, 30%, 40%, 50%,or greater than 50% as compared to an appropriate control value.

For example, if increased expression of a polypeptide desired, thenucleic acid may be an expression vector that includes a polynucleotidethat encodes the desired polypeptide. On the other hand, if reducedexpression of a polynucleotide or polypeptide is desired, then thenucleic acid may be, e.g., an antisense oligonucleotide, siRNA, ormicroRNA that comprises a polynucleotide sequence that specificallyhybridizes to a polynucleotide that encodes the target polypeptide,thereby disrupting expression of the target polynucleotide orpolypeptide. Alternatively, the nucleic acid may be a plasmid thatexpresses such an antisense oligonucleotide, siRNA, or microRNA.

In one particular embodiment, the invention provides a method ofmodulating the expression of a polypeptide by a cell, comprisingproviding to a cell a lipid particle that consists of or consistsessentially of a cationic lipid of formula I, a neutral lipid, a sterol,a PEG of PEG-modified lipid, e.g., in a molar ratio of about 20-65% ofcationic lipid of formula I, 3-25% of the neutral lipid, 15-55% of thesterol, and 0.5-15% of the PEG or PEG-modified lipid, wherein the lipidparticle is associated with a nucleic acid capable of modulating theexpression of the polypeptide. In particular embodiments, the molarlipid ratio is approximately 60/7.5/31/1.5, 57.5/7.5/31.5/3.5,57.2/7.1/34.3/1.4, 52/13/30/5, 50/10/38.5/1.5, 50/10/35/5, 40/10/40/10,40/15/40/5, or 35/15/40/10 (mol % cationic lipid of formula I/DSPC orDPPC/Chol/PEG-DMG or PEG-cDMA). In some embodiments, the lipid particlealso includes a targeting moiety such as a targeting lipid describedherein (e.g., the lipid particle consists essentially of a cationiclipid of formula I, a neutral lipid, a sterol, a PEG or PEG-modifiedlipid and a targeting moiety). In another group of embodiments, theneutral lipid in these compositions is replaced with DPPC, POPC, DOPE orSM. In another group of embodiments, the PEG or PEG-modified lipid isreplaced with PEG-DSG, PEG-DMG or PEG-DPG.

In particular embodiments, the therapeutic agent is selected from ansiRNA, a microRNA, an antisense oligonucleotide, and a plasmid capableof expressing an siRNA, a microRNA, or an antisense oligonucleotide, andwherein the siRNA, microRNA, or antisense RNA comprises a polynucleotidethat specifically binds to a polynucleotide that encodes thepolypeptide, or a complement thereof, such that the expression of thepolypeptide is reduced.

In other embodiments, the nucleic acid is a plasmid that encodes thepolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In related embodiments, the invention provides reagents useful fortransfection of cells in culture. For example, the lipid formulationsdescribed herein can be used to deliver nucleic acids to cultured cells(e.g., adherent cells, suspension cells, etc.).

In related embodiments, the invention provides a method of treating adisease or disorder characterized by overexpression of a polypeptide ina subject, comprising providing to the subject a pharmaceuticalcomposition of the invention, wherein the therapeutic agent is selectedfrom an siRNA, a microRNA, an antisense oligonucleotide, and a plasmidcapable of expressing an siRNA, a microRNA, or an antisenseoligonucleotide, and wherein the siRNA, microRNA, or antisense RNAcomprises a polynucleotide that specifically binds to a polynucleotidethat encodes the polypeptide, or a complement thereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of a cationic lipid offormula I, DSPC, Chol and PEG-DMG, PEG-C-DOMG or PEG-CDMA, e.g., in amolar ratio of about 20-65% of cationic lipid of formula I, 3-25% of theneutral lipid, 15-55% of the sterol, and 0.5-15% of the PEG orPEG-modified lipid PEG-DMG, PEG-C-DOMG or PEG-cDMA, wherein the lipidparticle is associated with the therapeutic nucleic acid. In particularembodiments, the molar lipid ratio is approximately 60/7.5/31/1.5,57.5/7.5/31.5/3.5, 57.2/7.1/34.3/1.4, 52/13/30/5, 50/10/38.5/1.5,50/10/35/5, 40/10/40/10, 35/15/40/10 or 40/15/40/5 (mol % cationic lipidof formula I/DSPC/Chol/PEG-DMG or PEG-cDMA). In some embodiments, thelipid particle also includes a targeting lipid described herein (e.g.,the lipid particle consists essentially of a cationic lipid of formulaI, a neutral lipid, a sterol, a PEG or PEG-modified lipid and atargeting moiety (e.g., GalNAc3-PEG-DSG)). In some embodiments, when thetargeting lipid includes a PEG moiety and is added to an existingliposomal formulation, the amount of PEG-modified lipid is reduced suchthat the total amount of PEG-modified lipid (i.e., PEG-modified lipid,for example PEG-DMG, and the PEG-containing targeting lipid) is kept ata constant mol percentage (e.g., 0.3%, 1.5 mol %, or 3.5 mol %). Inanother group of embodiments, the neutral lipid in these compositions isreplaced with DPPC, POPC, DOPE or SM. In another group of embodiments,the PEG or PEG-modified lipid is replaced with PEG-DSG or PEG-DPG. Inanother related embodiment, the invention includes a method of treatinga disease or disorder characterized by underexpression of a polypeptidein a subject, comprising providing to the subject a pharmaceuticalcomposition of the invention, wherein the therapeutic agent is a plasmidthat encodes the polypeptide or a functional variant or fragmentthereof.

The invention further provides a method of inducing an immune responsein a subject, comprising providing to the subject the pharmaceuticalcomposition of the invention, wherein the therapeutic agent is animmunostimulatory oligonucleotide. In certain embodiments, the immuneresponse is a humoral or mucosal immune response consists of or consistsessentially of a cationic lipid of formula I, DSPC, Chol and PEG-DMG,PEG-C-DOMG or PEG-cDMA, e.g., in a molar ratio of about 20-65% ofcationic lipid of formula I, 3-25% of the neutral lipid, 15-55% of thesterol, and 0.5-15% of the PEG or PEG-modified lipid PEG-DMG, PEG-C-DOMGor PEG-CDMA, wherein the lipid particle is associated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 60/7.5/31/1.5, 57.5/7.5/31.5/3.5,57.2/7.1/34.3/1.4, 52/13/30/5, 50/10/38.5/1.5, 50/10/35/5, 40/10/40/10,35/15/40/10 or 40/15/40/5 (mol % cationic lipid of formulaI/DSPC/Chol/PEG-DMG or PEG-cDMA). In some embodiments, the lipidparticle also includes a targeting lipid described herein (e.g., thelipid particle consists essentially of a cationic lipid of formula I, aneutral lipid, a sterol, a PEG or PEG-modified lipid and a targetingmoiety). In some embodiments, when the targeting lipid includes a PEGmoiety and is added to an existing liposomal formulation, the amount ofPEG-modified lipid is reduced such that the total amount of PEG-modifiedlipid (i.e., PEG-modified lipid, for example PEG-DMG, and thePEG-containing targeting lipid) is kept at a constant mol percentage(e.g., 0.3%, 1.5 mol %, or 3.5 mol %). In another group of embodiments,the neutral lipid in these compositions is replaced with DPPC, POPC,DOPE or SM. In another group of embodiments, the PEG or PEG-modifiedlipid is replaced with PEG-DSG or PEG-DPG. In further embodiments, thepharmaceutical composition is provided to the subject in combinationwith a vaccine or antigen. Thus, the invention itself provides vaccinescomprising a lipid particle of the invention, which comprises animmunostimulatory oligonucleotide, and is also associated with anantigen to which an immune response is desired. In particularembodiments, the antigen is a tumor antigen or is associated with aninfective agent, such as, e.g., a virus, bacteria, or parasite.

A variety of tumor antigens, infections agent antigens, and antigensassociated with other disease are well known in the art and examples ofthese are described in references cited herein. Examples of antigenssuitable for use in the invention include, but are not limited to,polypeptide antigens and DNA antigens. Specific examples of antigens areHepatitis A, Hepatitis B, small pox, polio, anthrax, influenza, typhus,tetanus, measles, rotavirus, diphtheria, pertussis, tuberculosis, andrubella antigens. In one embodiment, the antigen is a Hepatitis Brecombinant antigen. In other aspects, the antigen is a Hepatitis Arecombinant antigen. In another aspect, the antigen is a tumor antigen.Examples of such tumor-associated antigens are MUC-1, EBV antigen andantigens associated with Burkitt's lymphoma. In a further aspect, theantigen is a tyrosinase-related protein tumor antigen recombinantantigen. Those of skill in the art will know of other antigens suitablefor use in the invention.

Tumor-associated antigens suitable for use in the subject inventioninclude both mutated and non-mutated molecules that may be indicative ofsingle tumor type, shared among several types of tumors, and/orexclusively expressed or overexpressed in tumor cells in comparison withnormal cells. In addition to proteins and glycoproteins, tumor-specificpatterns of expression of carbohydrates, gangliosides, glycolipids andmucins have also been documented. Exemplary tumor-associated antigensfor use in the subject cancer vaccines include protein products ofoncogenes, tumor suppressor genes and other genes with mutations orrearrangements unique to tumor cells, reactivated embryonic geneproducts, oncofetal antigens, tissue-specific (but not tumor-specific)differentiation antigens, growth factor receptors, cell surfacecarbohydrate residues, foreign viral proteins and a number of other selfproteins.

Specific embodiments of tumor-associated antigens include, e.g., mutatedantigens such as the protein products of the Ras p21 protooncogenes,tumor suppressor p53 and BCR-abl oncogenes, as well as CDK4, MUM1,Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4,galectin 9, carbonic anhydrase, Aldolase A, PRAME, Her2/neu, ErbB-2 andKSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionicgonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA)and melanocyte differentiation antigens such as Mart 1/Melan A, gp100,gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such asPSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene productssuch as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and othercancer testis antigens such as NY-ESO1, SSX2 and SCP1; mucins such asMuc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutralglycolipids and glycoproteins such as Lewis (y) and globo-H; andglycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn.Also included as tumor-associated antigens herein are whole cell andtumor cell lysates as well as immunogenic portions thereof, as well asimmunoglobulin idiotypes expressed on monoclonal proliferations of Blymphocytes for use against B cell lymphomas.

Pathogens include, but are not limited to, infectious agents, e.g.,viruses, that infect mammals, and more particularly humans. Examples ofinfectious virus include, but are not limited to: Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 (also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g.,dengue viruses, encephalitis viruses, yellow fever viruses);Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g., vesicularstomatitis viruses, rabies viruses); Coronaviridae (e.g.,coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae(e.g., parainfluenza viruses, mumps virus, measles virus, respiratorysyncytial virus); Orthomyxoviridae (e.g., influenza viruses);Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae(e.g., African swine fever virus); and unclassified viruses (e.g., theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses).

Also, gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfuenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Additional examples of pathogens include, but are not limited to,infectious fungi that infect mammals, and more particularly humans.Examples of infectious fingi include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.Examples of infectious parasites include Plasmodium such as Plasmodiumfalciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.Other infectious organisms (i.e., protists) include Toxoplasma gondii.

Pharmaceutical Compositions

In one embodiment, the invention provides pharmaceutical compositionscomprising a nucleic acid agent identified by the liver screening modeldescribed herein. The composition includes the agent, e.g., a dsRNA, anda pharmaceutically acceptable carrier. The pharmaceutical composition isuseful for treating a disease or disorder associated with the expressionor activity of the gene. Such pharmaceutical compositions are formulatedbased on the mode of delivery. One example is compositions that areformulated for systemic administration via parenteral delivery.Pharmaceutical compositions including the identified agent areadministered in dosages sufficient to inhibit expression of the targetgene, e.g., the Factor VII gene. In general, a suitable dose of dsRNAagent will be in the range of 0.01 to 5.0 milligrams per kilogram bodyweight of the recipient per day, generally in the range of 1 microgramto 1 mg per kilogram body weight per day. The pharmaceutical compositionmay be administered once daily, or the dsRNA may be administered as two,three, or more sub-doses at appropriate intervals throughout the day oreven using continuous infusion or delivery through a controlled releaseformulation. In that case, the dsRNA contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation which providessustained release of the dsRNA over a several day period. Sustainedrelease formulations are well known in the art and are particularlyuseful for vaginal delivery of agents, such as could be used with theagents of the invention. In this embodiment, the dosage unit contains acorresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

In particular embodiments, pharmaceutical compositions comprising thelipid-nucleic acid particles of the invention are prepared according tostandard techniques and further comprise a pharmaceutically acceptablecarrier. Generally, normal saline will be employed as thepharmaceutically acceptable carrier. Other suitable carriers include,e.g., water, buffered water, 0.9% saline, 0.3% glycine, and the like,including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. In compositions comprising saline or othersalt containing carriers, the carrier is preferably added followinglipid particle formation. Thus, after the lipid-nucleic acidcompositions are formed, the compositions can be diluted intopharmaceutically acceptable carriers such as normal saline.

The resulting pharmaceutical preparations may be sterilized byconventional, well known sterilization techniques. The aqueous solutionscan then be packaged for use or filtered under aseptic conditions andlyophilized, the lyophilized preparation being combined with a sterileaqueous solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the lipidic suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as α-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The concentration of lipid particle or lipid-nucleic acid particle inthe pharmaceutical formulations can vary widely, i.e., from less thanabout 0.01%, usually at or at least about 0.05-5% to as much as 10 to30% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. For example, the concentration may be increasedto lower the fluid load associated with treatment. This may beparticularly desirable in patients having atherosclerosis-associatedcongestive heart failure or severe hypertension. Alternatively,complexes composed of irritating lipids may be diluted to lowconcentrations to lessen inflammation at the site of administration. Inone group of embodiments, the nucleic acid will have an attached labeland will be used for diagnosis (by indicating the presence ofcomplementary nucleic acid). In this instance, the amount of complexesadministered will depend upon the particular label used, the diseasestate being diagnosed and the judgement of the clinician but willgenerally be between about 0.01 and about 50 mg per kilogram of bodyweight (e.g., of the nucleic acid agent), preferably between about 0.1and about 5 mg/kg of body weight. In some embodiments a complexadministered includes from about 0.004 and about 50 mg per kilogram ofbody weight of nucleic acid agent (e.g., from about 0.006 mg/kg to about0.2 mg/kg).

As noted above, the lipid-therapeutic agent (e.g., nucleic acid)particles of the invention may include polyethylene glycol(PEG)-modified phospholipids, PEG-ceramide, or gangliosideG_(M1)-modified lipids or other lipids effective to prevent or limitaggregation. Addition of such components does not merely prevent complexaggregation. Rather, it may also provide a means for increasingcirculation lifetime and increasing the delivery of the lipid-nucleicacid composition to the target tissues.

The invention also provides lipid-therapeutic agent compositions in kitform. The kit will typically be comprised of a container that iscompartmentalized for holding the various elements of the kit. The kitwill contain the particles or pharmaceutical compositions of theinvention, preferably in dehydrated or concentrated form, withinstructions for their rehydration or dilution and administration. Incertain embodiments, the particles comprise the active agent, while inother embodiments, they do not.

The pharmaceutical compositions containing an agent identified by theliver screening model may be administered in a number of ways dependingupon whether local or systemic treatment is desired and upon the area tobe treated. Administration may be topical, pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Administration may also be designed to result inpreferential localization to particular tissues through local delivery,e.g. by direct intraarticular injection into joints, by rectaladministration for direct delivery to the gut and intestines, byintravaginal administration for delivery to the cervix and vagina, byintravitreal administration for delivery to the eye. Parenteraladministration includes intravenous, intraarterial, intraarticular,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the dsRNAs of the invention are in admixture with a topicaldelivery component, such as a lipid, liposome, fatty acid, fatty acidester, steroid, chelating agent or surfactant. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl ethanolamine(DOPE), dimyristoylphosphatidyl choline (DMPC), distearolyphosphatidylcholine) negative (e.g. dimyristoylphosphatidyl glycerol, or DMPG) andcationic (e.g. dioleoyltetramethylaminopropyl DOTAP anddioleoylphosphatidyl ethanolamine DOTMA). DsRNAs of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, dsRNAs may be complexedto lipids, in particular to cationic lipids. Preferred fatty acids andesters include but are not limited arachidonic acid, oleic acid,eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid,palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which dsRNAs of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsof the invention may be delivered orally, in granular form includingsprayed dried particles, or complexed to form micro or nanoparticles.DsRNA complexing agents include poly-amino acids; polyimines;polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S.application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, and liposome-containing formulations. These compositions maybe generated from a variety of components that include, but are notlimited to, preformed liquids, self-emulsifying solids andself-emulsifying semisolids.

The pharmaceutical formulations, which may conveniently be presented inunit dosage form, may be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thepharmaceutical carrier(s) or excipient(s). In general, the formulationsare prepared by uniformly and intimately bringing into association theactive ingredients with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

The compositions may be formulated into any of many possible dosageforms such as, but not limited to, tablets, capsules, gel capsules,liquid syrups, soft gels, suppositories, and enemas. The compositions ofthe invention may also be formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers.

In one embodiment of the invention the pharmaceutical compositions maybe formulated and used as foams. Pharmaceutical foams includeformulations such as, but not limited to, emulsions, microemulsions,creams, jellies and liposomes. While basically similar in nature theseformulations vary in the components and the consistency of the finalproduct. The preparation of such compositions and formulations isgenerally known to those skilled in the pharmaceutical and formulationarts and may be applied to the formulation of the compositions of theinvention

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description. The invention is capable of other embodiments andof being practiced or of being carried out in various ways. Also, thephraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing”, “involving”, andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

As used in the Examples provided herein, the term “ApoE” refers to ApoE3unless otherwise identified.

Example 1 siRNA Duplexes for Luc and FVII Targeting

Table 8 below provides exemplary sequences for the targeting of Luc andFVII.

TABLE 8 Sense/ Duplex Antisense Sequence 5′-3′ Target 1000/2434 CUU ACGCUG AGU ACU UCG Luc AdTdT U*CG AAG fUAC UCA GCG fUAA GdT*dT 2433/1001C*UfU ACG CUG AGfU ACU UCG Luc AdT*dT UCG AAG UAC UCA GCG UAA GdTdT2433/2434 C*UfU ACG CUG AGfU ACU UCG Luc AdT*dT U*CG AAG fUAC UCA GCGfUAA GdT*dT 1000/1001 CUU ACG CUG AGU ACU UCG Luc AdTdT UCG AAG UAC UCAGCG UAA GdTdT AD- GGAUCAUCUCAAGUCUUACdTdT FVII 1596GUAAGACUUGAGAUGAUCCdTdT AD- GGAfUfCAfUfCfUfCAAGfUfCfUfU FVII 1661AfCdTsdTGfUAAGAfCfUfUGAGAf UGAfUfCfCdT*dT Note:

lowercase is 2′-O-methyl modified nucleotide, * is phosphorothioatebackbone linkages, fN is a 2′-fluoro nucleotide, dN is 2′-deoxynucleotide.

Example 2 FVII In Vivo Evaluation Using the Cationic Lipid DerivedLiposomes

In Vivo Rodent Factor VII and ApoB Silencing Experiments.

C57BL/6 mice (Charles River Labs, MA) and Sprague-Dawley rats (CharlesRiver Labs, MA) received either saline or siRNA in desired formulationsvia tail vein injection at a volume of 0.01 mL/g. At various time pointspost-administration, animals were anesthetized by isofluorane inhalationand blood was collected into serum separator tubes by retro orbitalbleed. Serum levels of Factor VII protein were determined in samplesusing a chromogenic assay (Coaset Factor VII, DiaPharma Group, OH orBiophen FVII, Aniara Corporation, OH) according to manufacturerprotocols. A standard curve was generated using serum collected fromsaline treated animals. In experiments where liver mRNA levels wereassessed, at various time points post-administration, animals weresacrificed and livers were harvested and snap frozen in liquid nitrogen.Frozen liver tissue was ground into powder. Tissue lysates were preparedand liver mRNA levels of Factor VII and apoB were determined using abranched DNA assay (QuantiGene Assay, Panomics, Calif.).

Example 3 Liposome Formulations for FVII Targeting

Factor VII (FVII), a prominent protein in the coagulation cascade, issynthesized in the liver (hepatocytes) and secreted into the plasma.FVII levels in plasma can be determined by a simple, plate-basedcolorimetric assay. As such, FVII represents a convenient model fordetermining sirna-mediated downregulation of hepatocyte-derivedproteins, as well as monitoring plasma concentrations and tissuedistribution of the nucleic acid lipid particles and siRNA.

Factor VII Knockdown in Mice

FVII activity was evaluated in FVII siRNA-treated animals at 24 hoursafter intravenous (bolus) injection in C57BL/6 mice. FVII was measuredusing a commercially available kit for determining protein levels inserum or tissue, following the manufacturer's instructions at amicroplate scale. FVII reduction was determined against untreatedcontrol mice, and the results were expressed as % Residual FVII. Fourdose levels (2, 5, 12.5, 25 mg/kg FVII siRNA) were used in the initialscreen of each novel liposome composition, and this dosing was expandedin subsequent studies based on the results obtained in the initialscreen.

Determination of Tolerability

The tolerability of each novel liposomal siRNA formulation was evaluatedby monitoring weight change, cageside observations, clinical chemistryand, in some instances, hematology. Animal weights were recorded priorto treatment and at 24 hours after treatment. Data was recorded as %Change in Body Weight. In addition to body weight measurements, a fullclinical chemistry panel, including liver function markers, was obtainedat each dose level (2, 5, 12.5 and 25 mg/kg siRNA) at 24 hourspost-injection using an aliquot of the serum collected for FVIIanalysis. Samples were sent to the Central Laboratory for Veterinarians(Langley, BC) for analysis. In some instances, additional mice wereincluded in the treatment group to allow collection of whole blood forhematology analysis.

Determination of Therapeutic Index

Therapeutic index (TI) is an arbitrary parameter generated by comparingmeasures of toxicity and activity. For these studies, TI was determinedas:TI=MTD(maximum tolerated dose)/ED₅₀(dose for 50% FVII knockdown)

The MTD for these studies was set as the lowest dose causing >7%decrease in body weight and a >200-fold increase in alanineaminotransferase (ALT), a clinical chemistry marker with goodspecificity for liver damage in rodents. The ED₅₀ was determined fromFVII dose-activity curves.

AD 1661 siRNA as provided in Example 1 was administered in formulationscomprising the following molar ratio of DLin-M-C3-DMA:DSPC:Chol:PEG-DMG,which were prepared and tested in the methods as described in Example 2:60:7.5:31:1.5; 50:10:38:0.5:1.5; and 40:20:38.5:1.5. The results ofthese in vivo experiments are provided in FIG. 1, demonstrating thesilencing ability of the formulations as tested.

Example 4 Preparation of 1,2-Di-O-alkyl-sn3-Carbomoylglyceride (PEG-DMG)

Preparation of IVa

1,2-Di-O-tetradecyl-sn-glyceride Ia (30 g, 61.80 mmol) andN,N′-succinimidylcarbonate (DSC, 23.76 g, 1.5 eq) were taken indichloromethane (DCM, 500 mL) and stirred over an ice water mixture.Triethylamine (TEA, 25.30 mL, 3 eq) was added to the stirring solutionand subsequently the reaction mixture was allowed to stir overnight atambient temperature. Progress of the reaction was monitored by TLC. Thereaction mixture was diluted with DCM (400 mL) and the organic layer waswashed with water (2×500 mL), aqueous NaHCO₃ solution (500 mL) followedby standard work-up. The residue obtained was dried at ambienttemperature under high vacuum overnight. After drying the crudecarbonate IIa thus obtained was dissolved in dichloromethane (500 mL)and stirred over an ice bath. To the stirring solution mPEG₂₀₀₀-NH₂(III, 103.00 g, 47.20 mmol, purchased from NOF Corporation, Japan) andanhydrous pyridine (Py, 80 mL, excess) were added under argon. Thereaction mixture was then allowed to stir at ambient temperatureovernight. Solvents and volatiles were removed under vacuum and theresidue was dissolved in DCM (200 mL) and charged on a column of silicagel packed in ethyl acetate. The column was initially eluted with ethylacetate and subsequently with gradient of 5-10% methanol indichloromethane to afford the desired PEG-Lipid IVa as a white solid(105.30 g, 83%). ¹H NMR (CDCl₃, 400 MHz) δ=5.20-5.12 (m, 1H), 4.18-4.01(m, 2H), 3.80-3.70 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂),2.10-2.01 (m, 2H), 1.70-1.60 (m, 2H), 1.56-1.45 (m, 4H), 1.31-1.15 (m,48H), J=6.5 Hz, 6H). MS range found: 2660-2836.

Preparation of IVb

1,2-Di-O-hexadecyl-sn-glyceride Ib (1.00 g, 1.848 mmol) and DSC (0.710g, 1.5 eq) were taken together in dichloromethane (20 mL) and cooleddown to 0° C. in an ice water mixture. Triethylamine (1.00 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solutionand dried over sodium sulfate. Solvents were removed under reducedpressure and the resulting residue of IIb was maintained under highvacuum overnight. This compound was directly used for the next reactionwithout further purification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol,purchased from NOF Corporation, Japan) and IIb (0.702 g, 1.5 eq) weredissolved in dichloromethane (20 mL) under argon. The reaction wascooled to 0° C. Pyridine (1 mL, excess) was added and the reactionstirred overnight. The reaction was monitored by TLC. Solvents andvolatiles were removed under vacuum and the residue was purified bychromatography (first ethyl acetate followed by 5-10% MeOH/DCM as agradient elution) to obtain the required compound IVb as a white solid(1.46 g, 76%). ¹H NMR (CDCl₃, 400 MHz) δ=5.17 (t, J=5.5 Hz, 1H), 4.13(dd, J=4.00 Hz, 11.00 Hz, 1H), 4.05 (dd, J=5.00 Hz, 11.00 Hz, 1H),3.82-3.75 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.05-1.90 (m,2H), 1.80-1.70 (m, 2H), 1.61-1.45 (m, 6H), 1.35-1.17 (m, 56H), J=6.5 Hz,6H). MS range found: 2716-2892.

Preparation of IVc

1,2-Di-O-octadecyl-sn-glyceride Ic (4.00 g, 6.70 mmol) and DSC (2.58 g,1.5 eq) were taken together in dichloromethane (60 mL) and cooled downto 0° C. in an ice water mixture. Triethylamine (2.75 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solution,and dried over sodium sulfate. Solvents were removed under reducedpressure and the residue was maintained under high vacuum overnight.This compound was directly used for the next reaction without furtherpurification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol, purchased from NOFCorporation, Japan) and IIc (0.760 g, 1.5 eq) were dissolved indichloromethane (20 mL) under argon. The reaction was cooled to 0° C.Pyridine (1 mL, excess) was added and the reaction was stirredovernight. The reaction was monitored by TLC. Solvents and volatileswere removed under vacuum and the residue was purified by chromatography(ethyl acetate followed by 5-10% MeOH/DCM as a gradient elution) toobtain the desired compound IVc as a white solid (0.92 g, 48%). NMR(CDCl₃, 400 MHz) δ=5.22-5.15 (m, 1H), 4.16 (dd, J=4.00 Hz, 11.00 Hz,1H), 4.06 (dd, J=5.00 Hz, 11.00 Hz, 1H), 3.81-3.75 (m, 2H), 3.70-3.20(m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.80-1.70 (m, 2H), 1.60-1.48 (m, 4H),1.31-1.15 (m, 64H), 0.85 (t, J=6.5 Hz, 6H). MS range found: 2774-2948.

Example 5 Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate)

A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol(0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Compounds of the present invention can be synthesized by the proceduresdescribed in the following papers, which are hereby incorporated bytheir entirety:

-   1. Schlueter, Urs; Lu, Jun; Fraser-Reid, Bert. Synthetic Approaches    To Heavily Lipidated Phosphoglyceroinositides. Organic Letters    (2003), 5(3), 255-257.-   2. King, J. F.; Allbutt, A. D. Can. J. Chem. 1970, 48, 1754-1769-   3. Mach, Mateusz; Schlueter, Urs; Mathew, Felix; Fraser-Reid, Bert;    Hazen, Kevin C. Comparing n-pentenyl orthoesters and n-pentenyl    glycosides as alternative glycosyl donors. Tetrahedron (2002),    58(36), 7345-7354.

Example 6 Efficacy of MC3 Liposomes Having Various LiposomalCompositions in Rats

To examine the dose response of MC3 containing liposomal formulations inrats, the following liposomal formulations were prepared essentially asdescribed in Example 2. As provided in the table below, the components,included are indicated as follows: MC3-DSPC-Cholesterol.-PEG-C14. Table9 below provides exemplary formulations as tested.

Animals Sprague-Dawley Total 27 Inj Vol. (uL) 5 ul/g injection Conc. InjVol. Dose Group Group size Target siRNA (mg/mL) (uL/g) (mg/kg) Vehicle 13 5 PBS 2 3 FVII 1661 0.06  5 0.30 50-10-38.5-1.5 3 3 FVII 1661 0.02  50.10 50-10-38.5-1.5 4 3 FVII 1661 0.006 5 0.03 50-10-38.5-1.5 5 3 FVII1661 0.002 5 0.01 50-10-38.5-1.5 6 3 FVII 1661 0.06  5 0.30 40-15-40-5 73 FVII 1661 0.02  5 0.10 40-15-40-5 8 3 FVII 1661 0.006 5 0.0340-15-40-5 9 3 FVII 1661 0.002 5 0.01 40-15-40-5As shown in FIG. 2, the liposomal formulation having 50 mol % MC3 showeda dosage response curve with efficacy at slightly lower siRNAconcentrations than that of the liposomal formulation having 40 mol %MC3.

Example 7 Efficacy of MC1 Liposomes Show ApoE Dependence of in Mice

To further examine the role of ApoE in efficacy of various liposomeformulations, wildtype and ApoE knockout mice were administered MC3liposomes containing the AD-1661 siRNA composition, at 0.1, 0.03, and0.01 mg/kg essentially as described in Example 2. Half of the liposomeformulations were premixed with recombinant ApoE protein in order todetermine whether exogenous addition of ApoE can overcome the absence ofthe protein in mice.

Table 10 below shows exemplary formulations as tested.

TABLE 10 Experimental Plan Animals C57BL/6 and ApoE knockout Total 42Inj Vol. (uL) variable based on weight Group Conc. Dose Group size MouseType Target siRNA (mg/mL) (mg/kg) Vehicle 1 3 C57BL/6 0.00  PBS 2 3C57BL/6 FVII 1661 0.0100 0.100 MC3 50-10-38.5-1.5 w/ApoE 3 3 C57BL/6FVII 1661 0.0030 0.030 MC3 50-10-38.5-1.5 w/ApoE 4 3 C57BL/6 FVII 16610.0010 0.010 MC3 50-10-38.5-1.5 w/ApoE 5 3 C57BL/6 FVII 1661 0.01000.100 MC3 50-10-38.5-1.5 w/ApoE 6 3 C57BL/6 FVII 1661 0.0030 0.030 MC350-10-38.5-1.5 w/ApoE 7 3 C57BL/6 FVII 1661 0.0010 0.010 MC350-10-38.5-1.5 w/ApoE 8 3 ApoE knockout 0.00  PBS 9 3 ApoE knockout FVII1661 0.0100 0.100 MC3 50-10-38.5-1.5 w/ApoE 10 3 ApoE knockout FVII 16610.0030 0.030 MC3 50-10-38.5-1.5 w/ApoE 11 3 ApoE knockout FVII 16610.0010 0.010 MC3 50-10-38.5-1.5 w/ApoE 12 3 ApoE knockout FVII 16610.0100 0.100 MC3 50-10-38.5-1.5 w/ApoE 13 3 ApoE knockout FVII 16610.0030 0.030 MC3 50-10-38.5-1.5 w/ApoE 14 3 ApoE knockout FVII 16610.0100 0.010 MC3 50-10-38.5-1.5 w/ApoEFIG. 3 shows dose-dependent attenuation of FVII protein levels in wildtype (right bars) but not ApoE deficient knockout mice (left bars) whenadministered with the MC3-formulated liposomes, suggesting a role ofApoE in cellular uptake and/or delivery to the liver. MC3 liposomesformulated as described above with the 1661 siRNA were administered atconcentrations of 0.1, 0.03, and 0.01 mg/kg by itself or premixed withApoE lipoprotein. At much higher doses (e.g., ˜1.0 mg/kg or above),however, MC3-formulated formulations were found to mediate silencing ofthe FVII mRNA and protein (not shown). As shown in FIG. 3, MC3formulated liposomal formulations tested are unable to mediate silencingof FVII in ApoE knockout mice, unless pre-mixed with recombinant ApoE.Thus, activity could be rescued in ApoE knockout mice by premixing MC3(an MC3-containing liposome) with ApoE.

Example 8 Efficacy of MC3 Containing Liposomal Formulations Varying inMole Percentage and Tail Length of Phosphocholines

To examine the effect of variations on the mole percentage and taillength of phosphocholines on efficacy of various liposome formulations,various formulations comprising DSPC, DMPC and DLPC were tested forefficacy of FVII silencing at 0.01 or 0.03 mg/kg.

Table 11 below shows exemplary formulations as tested:

Experimental Plan Animals C57BL/6 Total 45 Inj Vol. (uL) variable basedon weight Group Conc. Dose Group size Target siRNA (mg/mL) (mg/kg)Vehicle 1 3 0.00  PBS 2 3 FVII 1661 0.0010 0.010 MC3 50-10-38.5-1.5 1661DSPC 3 3 FVII 1661 0.0003 0.003 MC3 50-10-38.5-1.5 1661 DSPC 4 3 FVII1661 0.0010 0.010 MC3 50-10-38.5-1.5 1661 DMPC 5 3 FVII 1661 0.00030.003 MC3 50-10-38.5-1.5 1661 DMPC 6 3 FVII 1661 0.0010 0.010 MC350-10-38.5-1.5 1661 DLPC 7 3 FVII 1661 0.0003 0.003 MC3 50-10-38.5-1.51661 DLPC 8 3 FVII 1661 0.0010 0.010 MC3 40-20-38.5-1.5 1661 DSPC 9 3FVII 1661 0.0003 0.003 MC3 40-20-38.5-1.5 1661 DSPC 10 3 FVII 16610.0010 0.010 MC3 40-20-38.5-1.5 1661 DMPC 11 3 FVII 1661 0.0003 0.003MC3 40-20-38.5-1.5 1661 DMPC 12 3 FVII 1661 0.0010 0.010 MC340-20-38.5-1.5 1661 DLPC 13 3 FVII 1661 0.0003 0.003 MC3 40-20-38.5-1.51661 DLPC 14 3 FVII 1661 0.0010 0.010 MC3 30-30-38.5-1.5 1661 DMPC 15 3FVII 1661 0.0003 0.003 MC3 30-30-38.5-1.5 1661 DMPCFIG. 4 shows the effects of changes in the mole percentage of the MC3,e.g., comparing 50 and 40 mole percent, and for the case of DMPCcontaining formulation, 50, 40, and 30 mole percent. FIG. 4 also showsthe effect of changes in the neutral lipid, showing the differingresults for MC3 liposomal formulations comprising DSPC, DMPC, and DLPC.

Example 9 Incorporation of GalNAc Lipids into Liposome Formulations

To explore potential alternate delivery mechanisms, in vivo experimentswere performed using liposome formulations comprising N-acetylgalactosamine (GalNAc) conjugated lipids. GalNAc was chosen as apossible targeting ligand as it is known that the GalNAc receptor isthought to be highly expressed in the liver. Studies were thereforeperformed in mice and rats to test the efficacy of the MC3 containingliposomal formulations further comprising the GalNAc3-PEG-DSG lipid ofFormula III essentially as described in Example 2. In all experiments,the total amount of PEG-conjugated lipids was kept constant (e.g., where0.5% mol of GalNAc3-PEG is added, the corresponding amount of PEG-DSGwas decreased by 0.5% mol). Four animals were used for each of the ninegroups per genotype in the experiment.

Table 12 below provides experimental detail for the methods includingMC3 containing liposomes having 5% PEG lipid concentration, where theformulations were tested in C57BL6 mice. The liposomes comprising thefollowing relative molar amounts: 50/10/35/5 of MC3/DSPC/Chol/PEG-DSG.Where 0.5% GalNAc3-PEG is added, the corresponding amount of PEG-DSG isreduced to 4.5%.

TABLE 12 Experimental Plan Animals C57BL6 Total 36 Inj Vol. (uL)variable based on weight Group Conc. Inj Vol. Dose Group size TargetsiRNA (mg/mL) (uL/g) (mg/kg) Vehicle 1 4 10 PBS 2 4 FVII 1661 0.1 101.00 50/10/35/5 3 4 FVII 1661 0.05 10 0.50 50/10/35/5 4 4 FVII 16610.025 10 0.25 50/10/35/5 5 4 FVII 1661 0.0125 10 0.125 50/10/35/5 6 4FVII 1661 0.1 10 1.00 50/10/35/4.5 w 0.5% GalNAc-lipid 7 4 FVII 16610.05 10 0.50 50/10/35/4.5 w 0.5% GalNAc-lipid 8 4 FVII 1661 0.025 100.25 50/10/35/4.5 w 0.5% GalNAc-lipid 9 4 FVII 1661 0.0125 10 0.12550/10/35/4.5 w 0.5% GalNAc-lipid

Table 13 below provides experimental detail for the methods includingMC3 containing liposomes having 10 mol % concentration of PEG-DSG lipid,where the formulations were tested in C57BL6 mice. The liposomescomprised the following relative molar amounts: 50/10/30/10 ofMC3/DSPC/Chol/PEG-DSG. Where 0.5% GalNAc3-PEG is added, thecorresponding amount of PEG-DSG is reduced to 9.5%.

TABLE 13 Experimental Plan Animals C57BL6 Total 36 Inj Vol. (uL)variable based on weight Group Conc. Inj Vol. Dose Group size TargetsiRNA (mg/mL) (uL/g) (mg/kg) Vehicle 1 4 10 PBS 2 4 FVII 1661 0.5 105.00 50/10/30/10 3 4 FVII 1661 0.25 10 2.50 50/10/30/10 4 4 FVII 16610.125 10 1.25 50/10/30/10 5 4 FVII 1661 0.0625 10 0.625 50/10/30/10 6 4FVII 1661 0.5 10 5 50/10/30/9.5 w 0.5% GalNAc-lipid 7 4 FVII 1661 0.2510 2.50 50/10/30/9.5 w 0.5% GalNAc 8 4 FVII 1661 0.125 10 1.2550/10/30/9.5 w 0.5% GalNAc 9 4 FVII 1661 0.0625 10 0.63 50/10/30/9.5 w0.5% GalNAcFIG. 5, shows the effects where increasing PEG-shielding decreasesnon-GalNAc mediated silences in C57BL6 mice. This is demonstrated withPEG concentrations of both 5% and 10% in C57BL6 mice. Inclusion ofC18-PEG (i.e., PEG-DSG) at 10 mol % effectively inhibits silencing,which can be overcome by substituting 0.5 mol % of the PEG lipid with anequimolar amount GalNAc-lipid (i.e., GalNAc3-PEG-DSG of Formula III).Therefore, increasing PEG-shielding (e.g., from 5 mol % to 10 mol %)appears to decrease non-GalNAc-mediated silencing, but also overallpotency.

Similar experiments were also performed in rats, wherein the PEG lipid(also PEG-DSG) was included in the liposomes at both 5 and 10 mole %.Table 14 below provides experimental detail for the methods includingMC3 containing liposomes having 5% PEG lipid concentration, where theformulations were tested in rats. The liposomes comprising the followingrelative molar amounts: 50/10/35/5 of MC3/DSPC/Chol/PEG-DSG. Where 0.5%GalNAc3-PEG is added, the corresponding amount of PEG-DSG is reduced to4.5%.

TABLE 14 Experimental Plan Animals Sprague-Dawley Rats Total 36 Inj Vol.(uL) Bolus injection Group Conc. Inj Vol. Dose Group size Target siRNA(mg/mL) (uL/g) (mg/kg) Vehicle 1 4 5 PBS 2 4 FVII 1661 0.2 5 1.0050/10/35/5 3 4 FVII 1661 0.1 5 0.50 50/10/35/5 4 4 FVII 1661 0.05 5 0.2550/10/35/5 5 4 FVII 1661 0.025 5 0.125 50/10/35/5 6 4 FVII 1661 0.2 51.00 50/10/35/4.5 w 0.5% GalNAc-lipid 7 4 FVII 1661 0.1 5 0.5050/10/35/4.5 w 0.5% GalNAc-lipid 8 4 FVII 1661 0.05 5 0.25 50/10/35/4.5w 0.5% GalNAc-lipid 9 4 FVII 1661 0.025 5 0.125 50/10/35/4.5 w 0.5%GalNAc-lipid

Table 15 below provides experimental detail for the methods includingMC3 containing liposomes having 10% PEG lipid concentration, where theformulations were tested in rats. The liposomes comprising the followingrelative molar amounts: 50/10/30/10 of MC3/DSPC/Chol/PEG-DSG. Where 0.5%GalNAc3-PEG is added, the corresponding amount of PEG-DSG is reduced to9.5%.

TABLE 15 Experimental Plan Animals Sprague-Dawley Total 36 Inj Vol. (uL)Bolus injection Group Conc. Inj Vol. Dose Group size Target siRNA(mg/mL) (uL/g) (mg/kg) Vehicle 1 4 5 PBS 2 4 FVII 1661 1 5 5.0050/10/30/10 3 4 FVII 1661 0.5 5 2.50 50/10/30/10 4 4 FVII 1661 0.25 51.25 50/10/30/10 5 4 FVII 1661 0.125 5 0.625 50/10/30/10 6 4 FVII 1661 15 5.00 50/10/30/9.5 w 0.5% GalNAc-lipid 7 4 FVII 1661 0.5 5 2.5050/10/30/9.5 w 0.5% GalNAc-lipid 8 4 FVII 1661 0.25 5 1.25 50/10/30/9.5w 0.5% GalNAc-lipid 9 4 FVII 1661 0.125 5 0.625 50/10/30/9.5 w 0.5%GalNAc-lipid

FIG. 6 shows results of MC3 formulations containing C18 PEG at 5 mol %and 10 mol % administered to rats at the indicated dosages. Formulationscontaining 10 mol % of PEG-DSG shows little silencing at theconcentrations tested (0.625-5 mg/kg) in rats. However, inclusion of 0.5mol % GalNAc3-PEG-DSG of Formula III (i.e., replacing 0.5 mol % of theC18-PEG), restores knockdown of FVII. Therefore, when compared withmice, in the rat, more highly shielded formulation generally betterretains potency as shown in the differences between concentrations of 5mol % and 10 mol % PEG.

Example 10 Evaluation of Variations of Mol % of Components in MC3Containing Liposomal Formulations with and without Inclusion of 0.5 Mol% GalNAc3-PEG-DSG

In order to determine the efficacy of MC3 containing liposomes havingdifferent mole percentage of components, with and withoutGalNAc3-PEG-DSG, the following liposomal formulations were prepared andtested in C57BL6 mice, substantially as described in Example 2 above.The components, as depicted in the table, are provided in the order asfollows: MC3/DSPC/Chol./PEG-DSG. Where 0.5% GalNAc3-PEG is added, thecorresponding amount of PEG-DSG is reduced to 4.5%, as shown in Table 16below.

TABLE 16 Animals C57BL6 Total 33 Inj Vol (uL) variable based on weightGroup Conc. Inj Vol. Dose Group size Target siRNA (mg/mL) (uL/g) (mg/kg)Vehicle 1 3 10 PBS 2 3 FVII 1661 0.1 10 1.00 50/10/35/5 3 3 FVII 16610.1 10 1.00 50/10/35/4.5 w 0.5% GalNAc-lipid 4 3 FVII 1661 0.1 10 1.0040/15/40/5 5 3 FVII 1661 0.1 10 1.00 40/15/40/4.5 w 0.5% GalNAc-lipid 63 FVII 1661 0.1 10 1.00 30/25/40/5 7 3 FVII 1661 0.1 10 1.0030/25/40/4.5 w 0.5% GalNAc-lipid 8 3 FVII 1661 0.1 10 1.00 20/35/40/5 93 FVII 1661 0.1 10 1.00 20/35/40/4.5 w 0.5% GalNAc-lipid

As shown in FIG. 7, addition of the GlaNAc to the liposomal formulationsimproves silencing of FVII in each formulation, i.e., wherein the MC3 ispresent at 50, 40, and 30 mol %.

Example 11 Efficacy of MC3 and GalNAc Containing Liposomes in WT andASGPR KO Mice

To examine the role of ASGPR in efficacy of various liposomeformulations, wildtype and ASGPR knockout mice were administered MC3liposomes containing the AD-1661 siRNA composition, at 3, 1, and 0.3mg/kg as described in Example 1. The components, as depicted in thetable, are provided in the order as follows: MC3/DSPC/Chol./PEG-DSG.Where 0.5% GalNAc3-PEG is added, the corresponding amount of PEG-DSG isreduced to 9.5%, as shown in Table 17 below.

TABLE 17 Experimental Plan Animals C57BL6 and ASGPr KO Total 25 + 15 InjVol (uL) variable based on weight Group Conc. Inj Vol. Dose Group sizeTarget siRNA (mg/mL) (uL/g) (mg/kg) Vehicle 1 5 10 PBS 2 5 FVII 1661 0.310 3.00 50/10/30/10 3 5 FVII 1661 0.3 10 3.00 50/10/30/9.5 w 0.5%GalNAc-lipid 4 5 FVII 1661 0.1 10 1.00 50/10/30/9.5 w 0.5% GalNAc-lipid5 5 FVII 1661 0.03 10 0.300 50/10/30/9.5 w 0.5% GalNAc-lipid 6 5 10 PBS7 5 FVII 1661 0.3 10 3.00 50/10/30/10 8 5 FVII 1661 0.3 10 3.0050/10/30/9.5 w 0.5% GalNAc-lipid

FIG. 8 shows the results of these experiments, demonstrating thatrestoration of FVII knockdown in formulations containing C18 PEG byinclusion of the GalNAc3-PEG-DSG lipid is abolished when administered ina mouse strain deficient in the Asialoglycoprotein Receptor (ASGPR),which is the expected receptor for GalNAc targeting moiety.

Example 12 Oligonucleotide Synthesis

Synthesis

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG, 500Å, Prime Synthesis) and RNA phosphoramidites with standard protectinggroups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-diisopropyl-2-cyanoethylphosphoramidite,O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxyirityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N⁴-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeare purchased from (Promega). All phosphoramidites are used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichis used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes is used. The activator is 5-ethyl thiotetrazole(0.75M, American International Chemicals); for the PO-oxidationiodine/water/pyridine is used and for the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence is performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled siRNAs are synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite are purchased from Biosearch Technologies. Conjugationof ligands to 5′-end and or internal position is achieved by usingappropriately protected ligand-phosphoramidite building block. Anextended 15 min coupling of 0.1 M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid-support-bound oligonucleotide. Oxidation of theinternucleotide phosphite to the phosphate is carried out using standardiodine-water as reported (1) or by treatment with ten-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate is introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent. The cholesterol phosphoramidite is synthesized in house andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite is 16 minutes.Deprotection I (Nucleobase Deprotection)After completion of synthesis, the support is transferred to a 100 mLglass bottle (VWR). The oligonucleotide is cleaved from the support withsimultaneous deprotection of base and phosphate groups with 80 mL of amixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C.The bottle is cooled briefly on ice and then the ethanolic ammoniamixture is filtered into a new 250-mL bottle. The CPG is washed with2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixtureis then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dryice and dried under vacuum on a speed vac.Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionis then quenched with 50 mL of 20 mM sodium acetate and the pH isadjusted to 6.5. Oligonucleotide is stored in a freezer untilpurification.

Analysis

The oligonucleotides are analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phasepreparative HPLC. The unconjugated oligonucleotides are purified byanion-exchange HPLC on a TSK gel column packed in house. The buffers are20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodiumphosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides are pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides arediluted in water to 150 μL and then pipetted into special vials for CGEand LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.siRNA PreparationFor the preparation of siRNA, equimolar amounts of sense and antisensestrand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to roomtemperature. Integrity of the duplex is confirmed by HPLC analysis.

TABLE 18  siRNA duplexes for Luc and FVII targeting Seq. Duplex IDSequence 5′-3′ Target 1000/1001 1 CUU ACG CUG AGU ACU UCG Luc AdTdT 2UCG AAG UAC UCA GCG UAA GdTdT AD-1955 3 cuuAcGcuGAGuAcuucGAdTsdT Luc 4UCGAAGuACUcAGCGuAAGdTsdT AD-1596 5 GGAUCAUCUCAAGUCUUACdTdT FVII 6GUAAGACUUGAGAUGAUCCdTdT AD-1661 7 GGAfUfCAfUfCfUfCAAGfUfCf FVIIUfUAfCdTsdT 8 GfUAAGAfCfUfUGAGAfUGAfUf CfCdTsdT

Lower case is 2′OMe modification and Nf is a 2′F modified nucleobase, dTis deoxythymidine, s is phosphothioate

Example 13 Synthesis of mPEG2000-1,2-Di-O-alkyl-sn3-carbomoylglyceride

The PEG-lipids, such as mPEG2000-1,2-Di-O-alkyl-sn3-carbomoylglyceridewere synthesized using the following procedures:

mPEG2000-1,2-Di-O-alkyl-sn3-carbomoylglyceride

Preparation of compound 4a (PEG-DMG)

1,2-Di-O-tetradecyl-sn-glyceride 1a (30 g, 61.80 mmol) andN,N′-succinimidylcarboante (DSC, 23.76 g, 1.5 eq) were taken indichloromethane (DCM, 500 mL) and stirred over an ice water mixture.Triethylamine (25.30 mL, 3 eq) was added to stirring solution andsubsequently the reaction mixture was allowed to stir overnight atambient temperature. Progress of the reaction was monitored by TLC. Thereaction mixture was diluted with DCM (400 mL) and the organic layer waswashed with water (2×500 mL), aqueous NaHCO₃ solution (500 mL) followedby standard work-up. Residue obtained was dried at ambient temperatureunder high vacuum overnight. After drying the crude carbonate 2a thusobtained was dissolved in dichloromethane (500 mL) and stirred over anice bath. To the stirring solution in PEG₂₀₀₀-NH₂ (3, 103.00 g, 47.20mmol, purchased from NOF Corporation, Japan) and anhydrous pyridine (80mL, excess) were added under argon. In some embodiments, themethoxy-(PEG)_(x)-amine has an x=from 45-49, preferably 47-49, and morepreferably 49. The reaction mixture was then allowed stir at ambienttemperature overnight. Solvents and volatiles were removed under vacuumand the residue was dissolved in DCM (200 mL) and charged on a column ofsilica gel packed in ethyl acetate. The column was initially eluted withethyl acetate and subsequently with gradient of 5-10% methanol indichloromethane to afford the desired PEG-Lipid 4a as a white solid(105.30 g, 83%). ¹H NMR (CDCl₃, 400 MHz) δ=5.20-5.12 (m, 1H), 4.18-4.01(m, 2H), 3.80-3.70 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂),2.10-2.01 (m, 2H), 1.70-1.60 (m, 2H), 1.56-1.45 (m, 4H), 1.31-1.15 (m,48H), 0.84 (t, J=6.5 Hz, 6H). MS range found: 2660-2836.

Preparation of 4b

1,2-Di-O-hexadecyl-sn-glyceride 1b (1.00 g, 1.848 mmol) and DSC (0.710g, 1.5 eq) were taken together in dichloromethane (20 mL) and cooleddown to 0° C. in an ice water mixture. Triethylamine (1.00 mL, 3 eq) wasadded to that and stirred overnight. The reaction was followed by TLC,diluted with DCM, washed with water (2 times), NaHCO₃ solution and driedover sodium sulfate. Solvents were removed under reduced pressure andthe residue 2b under high vacuum overnight. This compound was directlyused for the next reaction without further purification. MPEG₂₀₀₀-NH₂ 3(1.50 g, 0.687 mmol, purchased from NOF Corporation, Japan) and compoundfrom previous step 2b (0.702 g, 1.5 eq) were dissolved indichloromethane (20 mL) under argon. The reaction was cooled to 0° C.Pyridine (1 mL, excess) was added to that and stirred overnight. Thereaction was monitored by TLC. Solvents and volatiles were removed undervacuum and the residue was purified by chromatography (first Ethylacetate then 5-10% MeOH/DCM as a gradient elution) to get the requiredcompound 4b as white solid (1.46 g, 76%). ¹H NMR (CDCl₃, 400 MHz) δ=5.17(J=5.5 Hz, 1H), 4.13 (dd, J=4.00 Hz, 11.00 Hz, 1H), 4.05 (dd, J=5.00 Hz,11.00 Hz, 1H), 3.82-3.75 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂),2.05-1.90 (m, 2H), 1.80-1.70 (m, 2H), 1.61-1.45 (m, 6H), 1.35-1.17 (m,56H), 0.85 (t, J=6.5 Hz, 6H). MS range found: 2716-2892.

Preparation of 4c

1,2-Di-O-octadecyl-sn-glyceride 1c (4.00 g, 6.70 mmol) and DSC (2.58 g,1.5 eq) were taken together in dichloromethane (60 mL) and cooled downto 0° C. in an ice water mixture. Triethylamine (2.75 mL, 3 eq) wasadded to that and stirred overnight. The reaction was followed by TLC,diluted with DCM, washed with water (2 times), NaHCO₃ solution and driedover sodium sulfate. Solvents were removed under reduced pressure andthe residue under high vacuum overnight. This compound was directly usedfor the next reaction with further purification. MPEG₂₀₀₀-NH₂ 3 (1.50 g,0.687 mmol, purchased from NOF Corporation, Japan) and compound fromprevious step 2c (0.760 g, 1.5 eq) were dissolved in dichloromethane (20mL) under argon. The reaction was cooled to 0° C. Pyridine (1 mL,excess) was added to that and stirred overnight. The reaction wasmonitored by TLC. Solvents and volatiles were removed under vacuum andthe residue was purified by chromatography (first Ethyl acetate then5-10% MeOH/DCM as a gradient elution) to get the required compound 4 cas white solid (0.92 g, 48%). ¹H NMR (CDCl₃, 400 MHz) δ=5.22-5.15 (m,1H), 4.16 (dd, J=4.00 Hz, 11.00 Hz, 1H), 4.06 (dd, J=5.00 Hz, 11.00 Hz,1H), 3.81-3.75 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.80-1.70(m, 2H), 1.60-1.48 (m, 4H), 1.31-1.15 (m, 64H), 0.85 (t, J=6.5 Hz, 6H).MS range found: 2774-2948.

Example 14 General Protocol for the Extrusion Method

Lipids (cationic lipid of formula I, DSPC, cholesterol, DMG-PEG) aresolubilized and mixed in ethanol according to the desired molar ratio.Liposomes are formed by an ethanol injection method where mixed lipidsare added to sodium acetate buffer at pH 5.2. This results in thespontaneous formation of liposomes in 35% ethanol. The liposomes areextruded through a 0.08 μm polycarbonate membrane at least 2 times. Astock siRNA solution was prepared in sodium acetate and 35% ethanol andwas added to the liposome to load. The siRNA-liposome solution wasincubated at 37° C. for 30 min and, subsequently, diluted. Ethanol wasremoved and exchanged to PBS buffer by dialysis or tangential flowfiltration.

Example 15 General Protocol for the In-Line Mixing Method

Individual and separate stock solutions are prepared—one containinglipid and the other siRNA. Lipid stock containing cationic lipid offormula I, DSPC, cholesterol and PEG lipid is prepared by solubilized in90% ethanol. The remaining 10% is low pH citrate buffer. Theconcentration of the lipid stock is 4 mg/mL. The pH of this citratebuffer can range between pH 3-5, depending on the type of fusogeniclipid employed. The siRNA is also solubilized in citrate buffer at aconcentration of 4 mg/mL. For small scale, 5 mL of each stock solutionis prepared.

Stock solutions are completely clear and lipids must be completelysolubilized before combining with siRNA. Therefore stock solutions maybe heated to completely solubilize the lipids. The siRNAs used in theprocess may be unmodified oligonucleotides or modified and may beconjugated with lipophilic moieties such as cholesterol.

The individual stocks are combined by pumping each solution to aT-junction. A dual-head Watson-Marlow pump is used to simultaneouslycontrol the start and stop of the two streams. A 1.6 mm polypropylenetubing is further downsized to a 0.8 mm tubing in order to increase thelinear flow rate. The polypropylene line (ID=0.8 mm) are attached toeither side of a T-junction. The polypropylene T has a linear edge of1.6 mm for a resultant volume of 4.1 mm³. Each of the large ends (1.6mm) of polypropylene line is placed into test tubes containing eithersolubilized lipid stock or solubilized siRNA. After the T-junction asingle tubing is placed where the combined stream will emit. The tubingis then extending into a container with 2× volume of PBS. The PBS israpidly stirring. The flow rate for the pump is at a setting of 300 rpmor 110 mL/min. Ethanol is removed and exchanged for PBS by dialysis. Thelipid formulations are then concentrated using centrifugation ordiafiltration to an appropriate working concentration.

Example 16 Synthesis of[6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate](a cationic lipid of formula I or MC3)

Preparation of Alcohol 2

A clean, dry 200 L glass reactor fitted with an argon inlet andthermowell was charged with 60 L of THF and 5.73 Kg (20.4 mol) oflinoleic acid. The contents of the reactor were cooled below 0° C. usingan acetone-dry ice bath. To this cold solution 13.8 L of Vitride (60%wt/vol) in toluene was added slowly maintaining the internal temperatureof the reaction mixture below 0° C. (Note: Initial addition of vitridewas exothermic and frothing was observed. The frothing ceased after 15minutes of addition). The addition of vitride took 3 hr and 45 minutes.After completion of the addition, the reaction mixture was stirred atambient temperature for 2 hr. An aliquot was taken and quenched withsat. Na₂SO₄ and the thus obtained crude product was analyzed by TLC forthe presence of the starting acid. The TLC showed completion of thereaction and the reaction mixture was again cooled below 0° C. in about45 minutes. A saturated solution of sodium sulfate (prepared bydissolving 1.1 Kg of sodium sulfate in 1.5 L of water) was slowly addedto the reaction mixture over 45 min. After completion of the addition,25 L of ethyl acetate was added over a period of 30 min with stirring.The obtained reaction mixture was filtered through a celite bed over aperiod of 45 min and the celite bed was washed with an additional 17 Lof ethyl acetate to remove all product from the residue. The combinedorganics were concentrated under reduced pressure. The residue wasdissolved in 15 L of ethyl acetate and the organic layer was washed withwater (2×7 L) and dried over sodium sulfate (1.1 Kg). After filtrationthe organic layer was concentrated under reduced pressure and driedunder high vacuum to obtain the product linoleyl alcohol as an oil.Crude yield=5.5 Kg (theoretical yield=5.43 Kg). This product was usedwithout further purification in the next step.Process for Preparing Linoleyl Mesylate 3A clean, dry 200 L all glass reactor fitted with argon inlet andthermowell was charged with 45 L of DCM and 5.5 Kg of the crude productfrom step 1. To this solution 11.5 L triethylamine was added followed by0.252 Kg (2.0 mol) of DMAP. The solution was cooled to −10° C. using adry-ice acetone mixture and to this cold reaction mass, a solution ofmesyl chloride (3.2 L, 41.3 mol) in DCM (10 L) was added drop wise overa period of 3 hrs while maintaining the temperature below 0° C. Aftercompletion of the addition, the reaction mixture was stirred at 0° C.for 1 h after which the TLC (5% EtOAc in DCM; PMA stain) of the reactionmixture showed complete disappearance of starting alcohol. To thereaction mixture, 17 L of ice-cold water was added and the layers wereseparated. The top aqueous layer was again washed with 10 L of DCM andthe layers were separated. The combined organic layers were washed with2×10 L of dilute hydrochloric acid (prepared by mixing 2 L of Con. HClwith 18 L of RO water), 2×7.5 L of water and 10 L of brine (prepared bydissolving 11 Kg of NaCl in 10 L of RO water). The organic layer wasseparated, dried over Na₂SO₄ (2.75 Kg) and filtered. The organic layerwas evaporated under reduced pressure and vacuum dried to obtain thecrude mesylate as a light yellow oil. Crude yield=7.1 Kg (theoreticalyield=7.1 Kg). This material was used without further purification inthe next step. ¹H NMR (CDCl₃, 400 MHz) δ=5.42-5.21 (m, 4H), 4.20 (t,2H), 3.06 (s, 3H), 2.79 (t, 2H), 2.19-2.00 (m, 4H), 1.90-1.70 (m, 2H),1.06-1.18 (m, 18H), 0.88 (t, 3H). ¹³C NMR (CDCl₃) δ=130.76, 130.54,128.6, 128.4, 70.67, 37.9, 32.05, 30.12, 29.87, 29.85, 29.68, 29.65,29.53, 27.72, 27.71, 26.15, 25.94, 23.09, 14.60. MS. Molecular weightcalculated for C₁₉H₃₆O₃S, Cal. 344.53. Found 343.52 (M-H⁻).

Preparation of Linoleyl Bromide 4

A clean, dry 200 L all glass reactor fitted with argon inlet andthermowell was charged with 25 L of DMF and 7.1 Kg of the crude productfrom step 2. This mixture was cooled to −10° C. with acetone-dry-icemixture. To this stirred mixture, a solution of lithium bromide (2.7 Kg,31.0 mol) in 25 L of DMF was added over a period of 1.5 hrs whilemaintaining the reaction temperature below 0° C. After completion of theaddition, the reaction mixture was stirred at 45° C. for 18-20 h untilTLC (10% EtOAc in hexanes, PMA stain) of an aliquot showed completedisappearance of the starting mesylate. The reaction mixture was dilutedwith 70 L of water and extracted with 57 L of hexanes. The aqueous layerwas further extracted with 2×10 L of hexanes and the combined organiclayers (approximately 120 L) were washed again with 2×10 L of water and1×10 L of brine (prepared by dissolving 14 Kg of sodium chloride in 10 Lof water). The obtained organic layer (120 L) was dried over sodiumsulfate (4 Kg) and concentrated under reduced pressure to obtain thecrude product (6.5 Kg). The crude product was purified by columnchromatography using 60-120 mesh silica gel using hexanes as eluent.Concentration of the pure product provided 5.5 Kg (81%, three steps) ofthe bromide 4 as a colorless liquid. ¹H NMR (CDCl₃, 400 MHz) δ=5.41-5.29(m, 4H), 4.20 (d, 2H), 3.40 (t, J=7 Hz, 2H), 2.77 (t, J=6.6 Hz, 2H),2.09-2.02 (m, 4H), 1.88-1.00 (m, 2H), 1.46-1.27 (m, 18H), 0.88 (t, J=3.9Hz, 3H). ¹³C NMR (CDCl₃) δ=130.41, 130.25, 128.26, 128.12, 34.17, 33.05,31.75, 29.82, 29.57, 29.54, 29.39, 28.95, 28.38, 27.42, 27.40, 25.84,22.79, 14.28.

Preparation of Dilinoleylmethanol 6

A clean, dry 20 L all glass reactor fitted with argon inlet, refluxcondenser and thermowell was degassed and purged with argon. The reactorwas charged with 277 g (11.3 mol) of activated magnesium followed by 1.5L of anhydrous ether. The reactor was again degassed three times andpurged with argon. The bromide 4 (2.5 Kg, 7.6 mol) was dissolved in 5 Lof anhydrous ether under argon and 1 L of this solution was added to thereactor followed by 25 mL (0.35 mol) of dibromomethane. The contents ofthe reactor were heated to 40° C. using a water bath (effervescence wasobserved followed by reflux indicating the initiation of Grignardreagent formation). After the initiation of the reaction, the heatingwas removed from the reactor and the remaining 4 L of the bromide wasslowly added over a period of 2 hr 30 min maintaining a gentle reflux ofthe mixture. After completion of the addition, the reaction mixture wasagain heated to reflux (bath temperature 45° C.) for 1 hr after which analiquot of the reaction mixture was quenched with water and analyzed byTLC (Hexanes, PMA stain) which showed complete consumption of startingbromide. The reaction mixture was cooled below 10° C. using an ice bathand a solution of ethyl formate (275 mL in 4 L of ether) in ether wasadded over a period of 2 hr 30 min and after completion of the additionthe reaction mixture was warmed to room temperature and stirred for 1hr. The reaction mixture was cooled back to 10° C. and acetone (1:15 L)was added slowly to the mixture followed by the addition of 7 L ofice-cold water and a solution of 10% sulfuric acid (prepared by diluting3.4 L of sulfuric acid with 34 L of ice-cold water). The product wasextracted with 3×10 L of ether and the combined organic layers werewashed with 10 L of brine and dried over sodium sulfate (2 Kg).Concentration of the organic layer over reduced pressure provided thecrude product (2 Kg) as a mixture of required dilindleyl alcohol alongwith minor amounts of O-formylated product. This crude product wasredissoloved in THF (4 L) and charged into the 20 L glass reactor. Tothis a solution of NaOH (0.934 Kg dissolved in 8 L of ice-cold water)was added and the contents were heated at 65° C. for 18 h after whichthe TLC (10% ether in hexanes) showed complete conversion of theO-formylated product to the required dilinoleylmethanol. The reactionmixture was cooled and was extracted with ether (3×4 L) and the combinedorganic layers were washed with 5 L of brine and dried over sodiumsulfate (4 Kg). Filtration followed by concentration of the organiclayer provided the crude product. The thus obtained crude product waspurified by column chromatography using 60-120 mesh silica gel using 4%ether in hexanes. Concentration of the pure product fractions providedthe pure 6 (1.45 Kg, 80%) as a colorless liquid. NMR (400 MHz, CDCl₃) δ5.47-5.24 (m, 8H), 3.56 (dd, J=6.8, 4.2, 1H), 2.85-2.66 (m, 4H),2.12-1.91 (m, 9H), 1.50-1.17 (m, 46H), 0.98-0.76 (m, 6H). ¹³C NMR (101MHz, CDCl₃) δ 130.41, 130.37, 128.18, 128.15, 77.54, 77.22, 76.91,72.25, 37.73, 31.75, 29.94, 29.89, 29.83, 29.73, 29.58, 2953, 27.46,27.43, 25.89, 25.86, 22.80, 14.30.

Preparation of[6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate]MC3 (8)

The dilinoleyl methanol 6 (144 g, 272 mmol) was dissolved in 1 L ofdichloromethane and to it the hydrochloride salt of dimethylaminobutyricacid 7 (55 g, 328 mmol) was added followed by diisopropylethylamine (70mL) and DMAP (4 g). After stirring for 5 min. at ambient temperature,MC1 (80 g, 417 mmol) was added and the reaction mixture was stirred atroom temperature overnight after which the TLC (silica gel, 5% MeOH inCH₂Cl₂) analysis showed complete disappearance of the starting alcohol.The reaction mixture was diluted with CH₂Cl₂ (500 mL) and washed withsaturated NaHCO₃ (400 mL), water (400 mL) and brine (500 mL). Thecombined organic layers were dried over anhyd. Na₂SO₄ and solvents wereremoved in vacuo. The crude product (180 g) thus obtained was purifiedby Flash column chromatography [2.5 Kg silica gel, Using the followingeluents i) column packed with 6 L of 0.1% NEt₃ in DCM; after loading ii)4 L of 0.1% NEt₃ in DCM; iii) 16 L of 2% MeOH—98% of 0.1% NEt₃ in DCM;iv) 4 L of 2.5% MeOH—97.5% of 0.1% NEt₃ in DCM; v) 12 L of 3% MeOH—97%of 0.1% NEt₃ in DCM] to isolate the pure product 8 (MC3, 159 g, 91%) asa colorless oil. ¹H NMR (400 MHz, CDCl₃): δ 5.46-5.23 (m, 8H), 4.93-4.77(m, 1H), 2.83-2.66 (m, 4H), 2.37-2.22 (m, 4H), 2.20 (s, 6H), 2.10-1.96(m, 9H), 1.85-1.69 (m, 2H), 1.49 (d, J=5.4, 4H), 1.39-1.15 (m, 39H),0.95-0.75 (m, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 173.56, 130.38, 130.33,128.17, 128.14, 7754, 77.22, 76.90, 74.44, 59.17, 45.64, 34.36, 32.69,31.73, 29.87, 29.76, 29.74, 29.70, 29.56, 29.50, 27.44, 27.41, 25.84,25.55, 23.38, 22.78, 14.27. EI-MS (+ve): MW calc. for C₄₃H₇₉NO₂ (M⁺H)⁺:642.6. found: 642.6.

Example 17 siRNA Formulation Using Preformed Vesicles

Cationic lipid containing particles were made using the preformedvesicle method. Cationic lipid, DSPC, cholesterol and PEG-lipid weresolubilised in ethanol at a molar ratio of 40/10/40/10, respectively.The lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4)with mixing to a final ethanol and lipid concentration of 30% (vol/vol)and 6.1 mg/mL respectively and allowed to equilibrate at roomtemperature for 2 min before extrusion. The hydrated lipids wereextruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22°C. using a Lipex Extruder (Northern Lipids, Vancouver, BC) until avesicle diameter of 70-90 nm, as determined by Nicomp analysis, wasobtained. This generally required 1-3 passes. For some cationic lipidmixtures which did not form small vesicles hydrating the lipid mixturewith a lower pH buffer (50 mM citrate, pH 3) to protonate the phosphategroup on the DSPC headgroup helped form stable 70-90 nm vesicles.The FVII siRNA (solubilised in a 50 mM citrate, pH 4 aqueous solutioncontaining 30% ethanol) was added to the vesicles, pre-equilibrated to35° C., at a rate of ˜5 mL/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was achieved, the mixture wasincubated for a further 30 min at 35° C. to allow vesiclere-organization and encapsulation of the FVII siRNA. The ethanol wasthen removed and the external buffer replaced with PBS (155 mM NaCl, 3mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.5) by either dialysis or tangential flowdiafiltration. The final encapsulated siRNA-to-lipid ratio wasdetermined after removal of unencapsulated siRNA using size-exclusionspin columns or ion exchange spin columns. The dose response curveillustrating the % residual FVII again the dose (mg/kg) is illustratedin FIG. 9.

Example 18 pKa Determination of a Cationic Lipid of Formula I

The pKa of the cationic lipid of formula I was determined essentially asdescribed (Eastman et al 1992 Biochemistry 31:4262-4268) using thefluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS),which is non-fluorescent in water but becomes appreciably fluorescentwhen bound to membranes. Vesicles composed of cationiclipid/DSPC/CH/PEG-c-DOMG (40:10:40:10 mole ratio) were diluted to 0.1 mMin buffers (130 mM NaCl, 10 mM CH₃COONH₄, 10 mM MES, 10 mM HEPES) ofvarious pH's, ranging from 2 to 11. An aliquot of the TNS aqueoussolution (1 μM final) was added to the diluted vesicles and after a 30second equilibration period the fluorescent of the TNS-containingsolution was measured at excitation and emission wavelengths of 321 nmand 445 nm, respectively. The pKa of the cationic lipid-containingvesicles was determined by plotting the measured fluorescence againstthe pH of the solutions and fitting the data to a Sigmodial curve usingthe commercial graphing program IgorPro. The pKa titration curve for thecationic lipid of formula I is shown in FIG. 10.

What is claimed is:
 1. A method of making a cationic lipid of formula I,or a pharmaceutically acceptable salt thereof,

comprising reacting dilinoleyl methanol with dimethylaminobutyric acid,or a salt thereof, under conditions to provide the cationic lipid offormula I, or a pharmaceutically acceptable salt thereof.
 2. The methodof claim 1, wherein the dilinoleyl methanol is reacted with ahydrochloride salt of dimethylaminobutyric acid under conditions toprovide a compound of formula I, or a pharmaceutically acceptable saltthereof.
 3. The method of claim 1, wherein dilinoleyl methanol isreacted with a salt of dimethylaminobutyric acid under conditions thatinclude EDCI, DMAP and DIPEA.
 4. The method of claim 1, furthercomprising purifying the compound of formula I, or a pharmaceuticallyacceptable salt thereof.
 5. The method of claim 4, wherein the compoundof formula I, or a pharmaceutically acceptable salt thereof, is purifiedusing Flash column chromatography.
 6. The method of claim 1, furthercomprising providing the dilinoleyl methanol by converting linoleylbromide to dilinoleyl methanol.
 7. The method of claim 6, wherein theconverting is carried out by reacting linoleyl bromide with magnesiumfollowed by water to provide a resulting mixture that comprisesdilinoleyl methanol.
 8. The method of claim 7, further comprisingtreating the resulting mixture with a base to provide dilinoleylmethanol.
 9. The method of claim 8, further comprising purifying thedilinoleyl methanol.
 10. The method of claim 9, wherein the dilinoleylmethanol is purified using column chromatography.